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Abstract:

This invention relates to novel phosphate-modified nucleosides, and
methods for producing them, being useful for the prevention or treatment
of a viral infection in a mammal, and for preparing oligonucleotides by
DNA/RNA polymerase-dependent amplification, e.g. PCR.

Claims:

1. A phosphate-modified nucleoside represented by the structural formula
(I) ##STR00025## wherein B is a pyrimidine or purine base, or an
analogue thereof, optionally substituted with one or two substituents
independently selected from the group consisting of halogen, hydroxyl,
sulfhydryl, methyl, ethyl, amino and methylamino; R1 is H or OH;
R3 is hydrogen; W is O or S; and R2 is a group represented by
the structural formula IV: ##STR00026## wherein n is 0 Z is selected
from the group consisting of O, S, NH and NCH3; each of R4 and
R5 is independently selected from the group consisting of
(CH2)m--COOR6 and (CH2)m-imidazolyl; R6 is
H or C1-6 alkyl; m is 0 or 1; and dotted lines represent the point
of attachment of Z to the phosphorous atom of formula (I); or the
structural formula (I'): ##STR00027## wherein B is a pyrimidine or
purine base, or an analogue thereof, optionally substituted with one or
two substituents independently selected from the group consisting of
halogen, hydroxyl, sulfhydryl, methyl, ethyl, isopropyl, amino,
methylamino, ethylamino, trifluoromethyl and cyano; R1 is hydrogen
or hydroxyl; R3 is selected from the group consisting of hydrogen,
C1-6 alkyl, C3-6 cycloalkyl, aryl-C1-6alkyl and
2-cyanoethyl, wherein said C1-6 alkyl, C3-6 cycloalkyl or
aryl-C1-6 alkyl is optionally substituted with one or more,
preferably 1, 2 or 3, substituents independently selected from the group
consisting of halogen, hydroxyl, C1-6 alkoxy, trifluoromethyl,
trifluoromethoxy, nitro, cyano and amino; W is O or S; and R2 is a
group represented by the structural formula (VI) ##STR00028## wherein
dotted lines represent the point of attachment of Z to the phosphorous
atom of formula (I'); n is 0, 1 or 2; Z is selected from the group
consisting of O, S, NH and NCH3; and Ar is an aryl group; and
stereoisomers, enantiomers and pharmaceutically acceptable salts thereof,
provided that said phosphate-modified nucleoside is not:
N-5'-adenylylphosphoramidate L-aspartic acid 1,4-dimethyl ester,
N-5'-uridylylphosphoramidate L-aspartic acid 1,4-dimethyl ester,
N-5'-thimidylylphosphoramidate L-aspartic acid 1,4-dimethyl ester,
N-5'-adenylylphosphoramidate L-aspartic acid 1,4-dimethyl ester,
monoammonium salt, N-5'-guanylylphosphoramidate L-aspartic acid
1,4-dimethyl ester, monoammonium salt, N-5'-uridylylphosphoramidate
L-aspartic acid 1,4-dimethyl ester, monoammonium salt,
N-5'-thymidylylphosphoramidate L-aspartic acid 1,4-dimethyl ester,
monoammonium salt, N-5'-citidylylphosphoramidate L-aspartic acid disodium
salt, N-5'-uridylylphosphoramidate L-histidine
N-5'-uridylylphosphoramidate L-histidine 1-methyl ester,
N-5'-adenylylphosphoramidate L-histidine 1-methyl ester,
N-5'-cytidylylphosphoramidate L-histidine 1-methyl ester, or
N-5'-uridylylphosphoramidate L-aspartic acid.

2. The phosphate-modified nucleoside of claim 1, wherein R4 is COOH
and R5 is selected from the group consisting of CH2--COOH and
CH2-(3H-imidazol-4-yl).

4. The phosphate-modified nucleoside of claim 1, wherein said pyrimidine
analogue is represented by the structural formula (B): ##STR00029##
wherein R7 is selected from the group consisting of --OH, --SH,
--NH2, --NHCH3 and --NHC2H5; R8 is selected from
the group consisting of hydrogen, methyl, ethyl, isopropyl, amino,
ethylamino, trifluoromethyl, cyano and halogen; and X is CH or N.

5. The phosphate-modified nucleoside of claim 4, wherein R4 is COOH
and R5 is selected from the group consisting of CH2--COOH and
CH2-(3H-imidazol-4-yl).

6. The phosphate-modified nucleoside of claim 1, wherein said purine
analogue is represented by the structural formula (D): ##STR00030##
wherein R9 is selected from the group consisting of H, --OH, --SH,
--NH2, and --NHCH3; R10 is selected from the group
consisting of hydrogen, methyl, ethyl, hydroxyl, amino and halogen; and Y
is CH or N.

7. The phosphate-modified nucleoside of claim 6, wherein R4 is COOH
and R5 is selected from the group consisting of CH2--COOH and
CH2-(3H-imidazol-4-yl).

8. A method for preparing an oligonucleotide comprising
phosphate-modified nucleosides, comprising the step of incorporating at
least one of said phosphate-modified nucleosides into a DNA/RNA strand,
wherein said phosphate-modified nucleoside is according to claim 1.

9. The method of claim 8, wherein the oligonucleotide is prepared by
DNA/RNA polymerase-dependent amplification.

10. The method of claim 9, wherein said DNA/RNA polymerase-dependent
amplification is PCR.

11. The method of claim 8, wherein the oligonucleotide is prepared by
administering said phosphate-modified nucleosides to bacteriae comprising
a DNA/RNA polymerase.

12. The method of claim 9, wherein said polymerase is from a
microorganism or from bacterial or viral origin.

14. The method of claim 13, wherein said polymerase is HIV Reverse
Transcriptase.

15. A non-pharmaceutical composition comprising a phosphate-modified
nucleoside according to claim 1, an aqueous solution and optionally one
or more buffering agents.

16. A pharmaceutical composition comprising a therapeutically effective
amount of a phosphate-modified nucleoside according to claim 1, and one
or more pharmaceutically acceptable excipients.

17. A pharmaceutical composition according to claim 16, wherein said
therapeutically effective amount is a viral polymerase inhibiting amount.

18. A pharmaceutical composition according to claim 16, wherein said
therapeutically effective amount is a HIV Reverse Transcriptase
inhibiting amount.

19. A method of prevention or treatment of a viral infection in a mammal,
comprising the administration of a therapeutically effective amount of a
phosphate-modified nucleoside according to claim 1, optionally in
combination with one or more pharmaceutically acceptable excipients.

20. The method of claim 19, wherein said viral infection is a HIV
infection.

21. The method of claim 19, wherein said mammal is a human being.

22. The phosphate-modified nucleoside of claim 1, being represented by
the structural formula (I'), wherein Ar is a phenyl group optionally
substituted with one or more substituents independently selected from the
group consisting of halogen, amino, trifluoromethyl, hydroxyl,
sulfhydryl, nitro, (C1-C6) alkoxy, trifluoromethoxy, cyano and
(CH2)q--COOR, wherein R is hydrogen or (C1-C6) alkyl,
and q is 0, 1 or 2.

23. The phosphate-modified nucleoside of claim 1, being represented by
the structural formula (I'), wherein R3 is hydrogen and Ar is
1,2-dicarboxylphenyl or 1,3-dicarboxylphenyl.

Description:

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application is a continuation-in-part of International
Application No. PCT/EP2008/001764, filed Feb. 27, 2008, which was
published in English under PCT Article 21(2), and which claims the
benefit of British Patent Application No. 0703715.3, filed Feb. 27, 2007;
British Patent Application No. 0703722.9, filed Feb. 27, 2007; British
Patent Application No. 0718228.0, filed Sep. 17, 2007; and British Patent
Application No. 0718229.8, filed Sep. 17, 2007, the disclosures of which
are incorporated by reference in their entirety.

[0002] This application also claims the benefit of British Patent
Application No. 0907436.0, filed Apr. 30, 2009, the disclosure of which
is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] The present invention relates to novel phosphate-modified
nucleosides, such as amino acid phosphoramidate nucleosides. The present
invention also relates to the phosphate-modified nucleosides as
substrates for wild type and/or mutated DNA or RNA polymerases.

[0004] The present invention provides for the use of these novel
phosphate-modified nucleosides for the production of oligonucleotides
such as DNA or RNA and of polypeptides or proteins. The invention also
relates to the use of these phosphate-modified nucleosides for growing or
selecting specific micro-organisms, such as bacteria. The invention
further provides for the use of these novel phosphate-modified
nucleosides to treat or prevent viral infections and their use to
manufacture a medicine to treat or prevent viral infections, particularly
infections with viruses belonging to the HIV family.

[0005] The present invention furthermore relates to a method for the
production of oligonucleotides, peptides or proteins by using said
phosphate-modified nucleosides.

BACKGROUND OF THE INVENTION

[0006] There has been significant progress in the design and synthesis of
numerous nucleotide analogues bearing a modified nucleobase moiety or
unnatural sugar and that are substrates for polymerases. Modifications at
the phosphate moiety are introduced to increase the stability of a
nucleotide toward enzymatic degradation or to mask the phosphate negative
charge and facilitate its penetration into a cell. Common strategy in
nucleotide prodrug design is protecting a phosphate moiety with a labile
masking group. Removal of a masking group liberates a nucleoside
monophosphate entity to be transformed into a nucleoside triphosphate
(hereinafter referred as NTP), a substrate for intracellular enzymes.
However, even after removal of the masking group, phosphorylation and
activation of nucleoside monophosphate remains a problem due to substrate
specificity of cellular kinases. Therefore, design of a nucleotide
analogue that would allow bypassing the kinase activation pathway while
behaving as a direct polymerase substrate would be a considerable
challenge.

[0007] Treatment of certain viral infections has always been a challenging
task due to ability of some viruses to integrate into a host's genome.
Therefore, the viral enzymes that are critical for viral genome
replication and integration are regarded as the most effective targets
for the design of anti-viral agents.

[0008] A lot of attention has been given to studying mechanisms of action
of Human Immunodeficiency Virus (type 1) (HIV-1) and developing specific
inhibitors towards this very challenging and important target. One of the
enzymes that are essential for the HIV replication is HIV reverse
transcriptase (HIV RT). The function of this enzyme is to use a viral RNA
genome and a reverse transcriptase to synthesize a double stranded DNA
for integration into a host genome. Because this step is critical for the
propagation of the viral infection, HIV reverse transcriptase (RT) is an
excellent target for anti-viral treatment. Currently, two major classes
of RT inhibitors (RTIs) exist and are administered for treatment of HIV
infection. Non-nucleoside reverse transcriptase inhibitors (NNRTs) are a
group of compounds that act through the allosteric inhibition by binding
to a hydrophobic site, or a pocket in close proximity to the active site
of HIV RT. The other group of RTIs is represented by nucleoside reverse
transcriptase inhibitors (NRTIs) that bind directly to the active site
and interfere with the polymerization reaction and DNA synthesis.

[0009] Nucleoside reverse transcriptase inhibitors are designed to be
recognized as substrates for RT and incorporated into a growing strand
for further termination of chain elongation. Inhibition of reverse
transcriptase activity and chain termination by NRTIs is achieved by
introduction of structural modifications to the sugar moiety. The
elongation of the DNA strand by a polymerase requires a nucleophilic
attack of the 3'-OH group to the a phosphorus atom of an incoming
nucleotide. Therefore, nucleoside analogs that lack the 3'-OH group or
have it substituted with other functional groups (for instance, N3,
F, H) not capable of the nucleophilic attack and formation of
phosphodiester bond would act as chain terminators.

[0010] Termination of DNA or RNA synthesis with nucleoside analogues is a
common and one of the most efficient strategies in the treatment of viral
infections, regardless of various side effects and cell toxicity. The
therapeutically active form of a nucleoside analogue is a nucleoside
triphosphate. However, at the physiological pH nucleoside triphosphates
are negatively charged molecules and thus they can not penetrate cellular
membranes. Hence, RT inhibitors are usually administered as biologically
inactive free nucleosides or as monophosphate prodrugs where a phosphate
group is masked with a lipophilic group.

[0011] There are three steps of kinase-mediated activation of anti-viral
nucleosides. At first, transformation to a monophosphate derivative takes
place through the action of a cytoplasmic nucleoside kinase (for
instance, thymidine kinase and deoxycytidine kinase). Furthermore, a
nucleoside 5'-monophosphate kinase catalyzes the conversion of a
nucleoside monophosphate to a nucleoside diphosphate. Finally, a
diphosphate derivative is phosphorylated by a nucleoside 5'-diphosphate
kinase (NDK) to provide an anti-viral nucleoside analog in its activated
(phosphorylated) form. The efficiency of phosphorylation depends on
substrate specificity of kinases. For instance, in the case of the AZT
phosphorylation cascade, conversion from the nucleoside monophosphate to
the nucleoside diphosphate becomes a rate limiting step as thymidylate
kinase (TMPK) catalyzes this conversion significantly slower than in the
case of the natural substrate (TMP). The consequences of this
inefficiency are accumulation of AZTMP in the cytosol and decreased
therapeutic concentration of AZTTP, the activated nucleoside form.
However, it was determined that high levels of AZTMP have an inhibitory
effect on thymidylate kinase by competing with its natural substrate
(TMP) and resulting in reduced levels of TDP and TTP. Moreover, increased
levels of AZT and its phosphorylated derivatives also affect other
enzymes of the de novo dNTPs synthesis resulting in skewed natural
nucleotide concentrations.

[0012] Therefore, administration of free NRTIs, which often relies on
intracellular phosphorylation and activation, has significant drawbacks.
One of the possible solutions is a prodrug or pronucleotide approach. In
the prodrug approach, the monophosphate moiety is "masked" with a labile
functional group which also serves to facilitate passage of a "masked"
nucleotide inside the cell. Once inside the cell, a masking group is
removed either enzymatically or through chemical activation. Removal of
the masking group affords a free nucleoside monophosphate intracellularly
where it can be further phosphorylated by TMPK and NDK. Thus, although
the prodrug approach facilitates delivery of an inhibitory nucleoside
inside the cell and eliminates the need for initial phosphorylation by a
nucleoside kinase, phosphorylation by TMPK and NDK are still required.

[0013] Besides delivery and bio-distribution challenges, another drawback
that is often associated with anti-viral therapy is emergence of
resistant strains. In the case of HIV-1, the drug resistance is developed
by appearance of mutations that would allow HIV RT to discriminate NRTIs
for natural nucleotides or remove an incorporated unnatural nucleobase by
excision reactions. It has also been shown for herpes simplex virus (HSV)
that reduction in anti-herpetic activity of acyclovir, a drug activated
by thymidine kinase phosphorylation and commonly used for treatment of
HSV infections, is mostly associated with thymidine kinase dependent
resistance. Established strategies to manage acyclovir-resistant HSV
infections include administration of anti-viral drugs acting directly on
a viral DNA polymerase (foscarnet, cidifovir) or by modulating immune
response of a patient. However, the later approach is not always feasible
and the former one could worsen patient's condition since these
medications impose a significant level of toxicity.

[0014] Therefore, considering all aforementioned aspects of therapy
directed to inhibit viral polymerases and reverse transcriptases, a
nucleotide analogue that would not depend on activation by
nucleoside/nucleotide kinases whilst serving as a natural substrate
mimic, would be of a great interest. In particular, there is a need in
the art for the development of novel phosphate-modified nucleosides that
meet the requirements for successful polymerase recognition, including
good chelating properties and spatial features to form stable
enzyme-substrate complexes, and whereby their incorporation reaction into
oligonucleotides is not stalled.

SUMMARY OF THE INVENTION

[0015] The present invention provides novel phosphate-modified nucleosides
which can act as substrates of DNA- or RNA-polymerases and/or as
antiviral agents.

[0016] The present invention provides novel phosphate-modified nucleosides
that can be used as alternative (compared to natural NTPs or dNTPs)
substrates for DNA- or RNA-polymerases. In a particular embodiment, these
phosphate-modified nucleotides are such that the pyrophosphate group of
nucleosides/nucleotides is replaced by a good leaving group, more
particularly a leaving group in a nucleotidyl transfer mechanism. In a
specific embodiment of the present invention, this leaving group is an
amino acid coupled by a phosphoramide binding, yet more particularly this
amino acid may be Asp (aspartic acid) or His (histidine), or a close
variant thereof, as defined below. In another specific embodiment of the
present invention, this leaving group is a carboxylic acid containing
group coupled by a phosphoramide binding moiety.

DETAILED DESCRIPTION OF THE INVENTION

[0017] A first aspect of this invention relates to novel
phosphate-modified nucleosides.

[0018] According to one first broad embodiment, the present invention
encompasses phosphate-modified nucleosides of (i.e. represented by) the
general structural formula A below:

##STR00001##

wherein [0019] Nuc is a natural nucleoside or a nucleoside analogue,
wherein said natural nucleoside or nucleoside analogue can be substituted
or unsubstituted, thereby creating a phosphonate or phosphate comprising
compound; [0020] R3 is independently selected from H (hydrogen);
(C1-C6) alkyl; (C3-C6) cycloalkyl;
aryl-(C1-C6) alkyl; and 2-cyanoethyl; wherein any such alkyl,
cycloalkyl or arylalkyl group may optionally be substituted with 1, 2 or
3 halogen, OH, (C1-C6)alkoxy, trifluoromethyl,
trifluoromethoxy, nitro, cyano, or amino substituents; [0021] W is
independently selected from O or S; and [0022] R2 is a group
represented by the structural formula V

##STR00002##

[0022] wherein [0023] n is 0 or 1; [0024] Z is selected from O; S; or
NH; [0025] each of R4 and R5 are independently selected from
(CH2)m--COOR6 and (CH2)m-imidazolyl; [0026]
R6 is selected from H (hydrogen) or (C1-C6) alkyl; [0027]
m is selected from 0, 1, and 2, and stereoisomers, enantiomers,
pharmaceutically acceptable salts and pro-drugs thereof.

[0028] According to a more specific embodiment of this invention, said
natural Nucleoside (Nuc) is coupled via its 5' position to the
phosphorous atom P in the structural formula A.

[0029] Another embodiment of the present invention relates to
phosphate-modified nucleotides as defined with reference to the
structural formula (I):

##STR00003##

wherein [0030] B is a pyrimidine or purine base, or an analogue
thereof, optionally substituted with one or two substituents
independently selected from the group consisting of halogen, hydroxyl,
sulfhydryl, methyl, ethyl, isopropyl, amino, methylamino, ethylamino,
trifluoromethyl and cyano; [0031] R1 is H or OH; [0032] R3 is
selected from the group consisting of H, C1-6 alkyl, C3-6
cycloalkyl, aryl-C1-6alkyl and 2-cyanoethyl, wherein said C1-6
alkyl, C3-6 cycloalkyl or aryl-C1-6 alkyl is optionally
substituted with one or more, preferably 1, 2 or 3, substituents
independently selected from the group consisting of halogen, OH,
C1-6 alkoxy, trifluoromethyl, trifluoromethoxy, nitro, cyano and
amino; [0033] W is O or S; and [0034] R2 is a group represented by
the structural formula IV:

[0054] In the structural formula (I), W is preferably O (oxygen) but it
can be replaced by S (sulfur) by chemical reactions well known in the
art. According to another embodiment of the present invention, the
molecular weight of the group R2 is not above 500.

[0055] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides of the structural formula (I) wherein
R1, R2, R3 and W have any of the values or meanings as
described herein, and wherein B is adenine; guanine; cytosine; thymine;
uracil, or a substituted uracil as described below.

[0056] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides of the structural formula (I) wherein
B, R1, R2 and W have any of the values or meanings as described
herein, and wherein R3 is H.

[0057] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides of the structural formula (I) wherein
B, R1, R2 and R3 have any of the values or meanings as
described herein, and wherein W is O.

[0058] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides of the structural formula (I) wherein
B, R2, R3 and W have any of the values or meanings as described
herein, and wherein R1 is H; and in another particular embodiment,
the present invention also relates to the phosphate-modified nucleoside
of formula I wherein B, R2, R3 and W have any of the values as
described herein, and wherein R1 is OH.

[0059] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides of the structural formula (I) wherein
B, R1, R2, R3 and W have any of the values or meanings
described herein, and wherein n is 0.

[0060] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides of the structural formula (I) wherein
R1, R2, R3 and W have any of the values or meanings
described herein, and wherein B is a pyrimidine or purine base analogue
as described in the Definitions section below, in particular
5-azapyrimidine, 5-azacytosine, 7-deazapurine, 7-deazaadenine,
7-deazaguanine, or 7-deaza-8-azapurines.

[0061] In a particular embodiment of the foregoing, the present invention
also relates to the phosphate-modified nucleoside of the structural
formula (I) wherein B, R1, R3 and W have any of the values or
meanings as described herein, and wherein R2 is a nitrogen-linked
natural or synthetic amino acid; and in another particular embodiment,
said amino acid is linked via its α-NH2 group. In yet another
particular embodiment, said amino acid is linked via its α-NH2
group, whereby the H group of the NH-linking group is substituted by a
methyl group.

[0062] In a more particular embodiment of the invention, said amino acid
is selected from Asp or His. In another particular embodiment of the
invention, said amino acid is selected from the group consisting of
L-alanine, D-alanine, beta-alanine and N-methyl beta-alanine
(3-(methylamino)propionic acid).

[0063] In yet another particular embodiment of the invention, said amino
acid is in the L conformation.

[0064] In another particular embodiment of the invention, with reference
to formula (IV), Z is O; NH or NCH3.

[0065] In yet another particular embodiment of the invention, R4 is
COOH and R5 is selected from CH2--COOH or
CH2-4-imidazolyl.

[0066] In a yet more particular embodiment of the invention, R3 is H
and R2 is according to formula IV, wherein Z is NH or NCH3,
R4 is COOH and R5 is CH2--COOH or CH2-4-imidazolyl.

[0067] In yet another particular embodiment, the present invention relates
to phosphate-modified nucleosides according to the structural formula
(I), wherein R3 is H and R2 is an amino acid coupled to the
phosphor atom through its amino function, more particularly said amino
acid is Aspartic acid (Asp), Histidine (His) or methyl-aspartic acid.

[0072] According to a second broad embodiment, the present invention
encompasses phosphate-modified nucleosides represented by the structural
formula A'

##STR00005##

wherein [0073] Nuc is a natural nucleoside or a nucleoside analogue,
wherein said natural nucleoside or nucleoside analogue can be substituted
or unsubstituted, thereby creating a phosphonate or phosphate comprising
compound; [0074] R3 is selected from the group consisting of
hydrogen, (C1-C6) alkyl, (C3-C6) cycloalkyl,
aryl-(C1-C6) alkyl, and 2-cyanoethyl; wherein any of such
alkyl, cycloalkyl or arylalkyl may optionally be substituted with 1, 2 or
3 substituents independently selected from the group consisting of
halogen, hydroxyl, (C1-C6) alkoxy, trifluoromethyl,
trifluoromethoxy, nitro, cyano and amino; [0075] W is O or S; [0076]
R2 is a group represented by the structural formula (VI)

##STR00006##

[0076] wherein [0077] dotted lines represent the point of attachment of
Z to the phosphorous atom of formula A'; [0078] n is 0, 1 or 2; [0079] Z
is selected from the group consisting of O, S, NH and NCH3; and
[0080] Ar is an aryl group such as defined below, and stereoisomers,
enantiomers, pharmaceutically acceptable salts and pro-drugs thereof.

[0081] According to a more specific embodiment of this invention, said
natural Nucleoside (Nuc) is coupled via its 5' position to the phosphor
atom P in formula A'.

[0082] Another embodiment of the present invention relates to
phosphate-modified nucleosides with reference to the structural formula
(I'):

##STR00007##

wherein [0083] B is a pyrimidine or purine base, or an analogue
thereof, optionally substituted with one or two substituents
independently selected from the group consisting of halogen, hydroxyl,
sulfhydryl, methyl, ethyl, isopropyl, amino, methylamino, ethylamino,
trifluoromethyl and cyano; [0084] R1 is hydrogen or hydroxyl; [0085]
R3 is selected from the group consisting of hydrogen, C1-6
alkyl, C3-6 cycloalkyl, aryl-C1-6alkyl and 2-cyanoethyl,
wherein said C1-6 alkyl, C3-6 cycloalkyl or aryl-C1-6
alkyl is optionally substituted with one or more, preferably 1, 2 or 3,
substituents independently selected from the group consisting of halogen,
hydroxyl, C1-6 alkoxy, trifluoromethyl, trifluoromethoxy, nitro,
cyano and amino; [0086] W is O or S; and [0087] R2 is a group
represented by the structural formula (VI)

##STR00008##

[0087] wherein [0088] dotted lines represent the point of attachment of
Z to the phosphorous atom of formula (I'); [0089] n is 0, 1 or 2; [0090]
Z is selected from the group consisting of O, S, NH and NCH3; and
[0091] Ar is an aryl group such as defined below, and stereoisomers,
enantiomers, pharmaceutically acceptable salts and pro-drugs thereof.

[0092] In the structural formulae (A') and (I'), W is preferably O but it
can be replaced by S by chemical reactions well known in the art.
According to a specific embodiment of the invention, the molecular weight
of R2 is not above 500.

[0093] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides represented by the structural formula
(I') wherein R1, R2, R3 and W may have any of the meanings
described herein, and wherein B is adenine, guanine, cytosine, thymine,
uracil, or a substituted uracil as described below.

[0094] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides represented by the structural formula
(I') wherein B, R1, R2 and W may have any of the meanings
described herein, and wherein R3 is hydrogen.

[0095] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides represented by the structural formula
(I') wherein B, R1, R2 and R3 may have any of the meanings
described herein, and wherein W is O.

[0096] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides represented by the structural formula
(I') wherein B, R2, R3 and W may have any of the meanings
described herein, and wherein R1 is hydrogen. In another particular
embodiment, the present invention also relates to the phosphate-modified
nucleoside of formula (I') wherein B, R2, R3 and W have any of
the meanings described herein, and wherein R1 is hydroxyl.

[0097] In a particular embodiment, the present invention relates to the
phosphate-modified nucleosides represented by the structural formula (I')
wherein B, R1, R2, R3 and W may have any of the meanings
described herein, and wherein n is 1.

[0098] In a particular embodiment, the present invention also relates to
the phosphate-modified nucleosides represented by the structural formula
(I') wherein R1, R2, R3 and W may have any of the meanings
described herein, and wherein B is any pyrimidine or purine base analogue
as defined below, in particular 5-azapyrimidine, 5-azacytosine,
7-deazapurine, 7-deazaadenine, 7-deazaguanine or 7-deaza-8-azapurine.

[0099] In another particular embodiment of the invention, with reference
to the structural formula (VI), Z may be O, NH or NCH3.

[0100] In another particular embodiment of the invention, with reference
to the structural formula (VI), aryl is a phenyl group optionally
substituted with one or more substituents independently selected from the
group consisting of halogen, amino, trifluoromethyl, hydroxyl,
sulfhydryl, nitro, (C1-C6) alkoxy, trifluoromethoxy, cyano and
(CH2)q--COOR, wherein R is hydrogen or (C1-C6) alkyl,
and q is 0, 1 or 2. In a more particular embodiment of the foregoing,
said phenyl is substituted with 1, 2, or 3 (CH2)q--COOR,
wherein R is hydrogen or (C1-C6)alkyl, and q is 0, 1 or 2.

[0101] In another particular embodiment of the invention, R3 is
hydrogen and aryl is a phenyl substituted with 1, 2, or 3
(CH2)q--COOR, wherein R is hydrogen or (C1-C6) alkyl,
and q is 0, 1 or 2.

[0102] In a yet more particular embodiment of the invention, R3 is
hydrogen and aryl is a phenyl substituted with two carboxylic acid
groups, such as 1,2-dicarboxylphenyl or 1,3-dicarboxylphenyl.

[0103] In a yet more particular embodiment of the invention, R3 is
hydrogen and Ar is 1,2-dicarboxylphenyl or 1,3-dicarboxylphenyl.

[0104] In a particular embodiment of the foregoing, the present invention
also relates to the phosphate-modified nucleoside represented by the
structural formula (I') wherein the pyrimidine analogue B is represented
by the structural formula (C):

##STR00009##

wherein [0105] R7 is selected from the group consisting of --OH,
--SH, --NH2, --NHCH3 and --NHC2H5; [0106] R8 is
selected from the group consisting of hydrogen, methyl, ethyl, isopropyl,
amino, ethylamino, trifluoromethyl, cyano and halogen; and [0107] X is CH
or N.

[0108] In yet another particular embodiment of the foregoing, the present
invention also relates to phosphate-modified nucleosides of formula (I')
wherein the purine analogue B is represented by the structural formula
(D):

##STR00010##

wherein [0109] R9 is selected from the group consisting of
hydrogen, --OH, --SH, --NH2, and --NHCH3; [0110] R10 is
selected from the group consisting of hydrogen, methyl, ethyl, hydroxyl,
amino and halogen; and [0111] Y is CH or N.

[0114] A second aspect of the first and second broad embodiments of the
present invention relates to the use of the phosphate-modified
nucleosides represented by the structural formulae (I), (I'), (A) and
(A'), including any of the above-referred specific embodiments thereof,
as a substrate for DNA- or RNA-polymerases, these polymerases being
either wild-type (naturally occurring) or mutated according to common
knowledge in the art. In a particular embodiment of the present
invention, said DNA- or RNA-polymerases may be selected from Therminator
DNA polymerase, KF (exo) DNA polymerase, or Reverse Transcriptase (e.g.
HIV-RT) or mutated forms of these enzymes. If needed, the enzymes as
described herein above can be mutated, using common knowledge in the art,
in order to better adapt to the novel phosphate-modified nucleosides
disclosed in this invention. Within this second aspect, the invention
relates to a method for preparing an oligonucleotide comprising
phosphate-modified nucleosides, comprising the step of incorporating at
least one of said phosphate-modified nucleosides into a DNA/RNA strand,
wherein said phosphate-modified nucleoside is represented by any one of
the structural formulae (A), (I), (A') and (I'), including any one of the
specific embodiments described herein.

[0115] Within the general framework of such a method, one or more of the
following embodiments may be particularly useful: [0116] the
oligonucleotide may be prepared by DNA/RNA polymerase-dependent
amplification; [0117] said DNA/RNA polymerase-dependent amplification is
PCR; [0118] the oligonucleotide may be prepared by administering said
phosphate-modified nucleosides to bacteriae comprising a DNA/RNA
polymerase; [0119] said polymerase may be from a microorganism or from
bacterial or viral origin; [0120] said polymerase may be Therminator DNA
polymerase; KF (exo.sup.-) DNA polymerase or Reverse Transcriptase.

[0121] In a particular embodiment, the phosphate-modified nucleosides of
this invention being represented by the structural formulae (I), (I'),
(A) and (A'), including any of the above-referred specific embodiments
thereof, can be used to build in at least 1, 2 or 3 nucleotides in a
growing DNA- or RNA-strand.

[0122] In another particular embodiment, the phosphate-modified
nucleosides of this invention being represented by the structural
formulae (I), (I'), (A) and (A'), including any of the above-referred
specific embodiments thereof, can be used to build in at maximum 1, 2 or
3 nucleotides in a growing DNA- or RNA-strand.

[0123] In another particular embodiment, the phosphate-modified
nucleosides of this invention being represented by the structural
formulae (I), (I'), (A) and (A'), including any of the above-referred
specific embodiments thereof, can be used to build in at most 300
nucleotides in a growing DNA- or RNA-strand.

[0124] In yet another particular embodiment, the phosphate-modified
nucleosides of this invention being represented by the structural
formulae (I), (I'), (A) and (A'), including any one of the above-referred
specific embodiments thereof, can be used in combination with a mixture
of natural dNTPs or NTPs (ATP,CTP,GTP,UTP,TTP) as a substrate for
DNA/RNA-polymerases, more in particular to built in 1-300 (e.g. 2-300)
nucleotides in a growing DNA- or RNA-strand.

[0125] The present invention also relates to the use of the
phosphate-modified nucleosides represented by the structural formulae
(I), (I'), (A) and (A'), including any one of the above-referred specific
embodiments thereof, for the enzymatic production of oligonucleotides,
peptides or proteins.

[0126] In a particular embodiment, the phosphate-modified nucleosides of
the invention being represented by the structural formulae (I), (I'), (A)
and (A'), including any one of the above-referred specific embodiments
thereof, can be used for in vitro production of DNA or RNA. In another
particular embodiment, the phosphate-modified nucleosides of the
invention being represented by the structural formulae (I), (I'), (A) and
(A'), including any one of the above-referred specific embodiments
thereof, can also be used for in vitro production of peptides or
proteins. In another particular embodiment the phosphate-modified
nucleosides of the invention being represented by any one of the
structural formulae (I), (I'), (A) and (A'), including any one of the
above-referred specific embodiments thereof, can be used for PCR
(polymerase chain reaction).

[0127] In yet another particular embodiment, the phosphate-modified
nucleosides of the invention being represented by any one of the
structural formulae (I), (I'), (A) and (A'), including any one of the
above-referred specific embodiments thereof, can be used as a substrate
for the growth of wild type and/or mutated bacteriae. In a particular
embodiment, the phosphate-modified nucleosides of the invention being
represented by any one of the structural formulae (I), (I'), (A) and
(A'), including any one of the above-referred specific embodiments
thereof, can be used as a substrate for the growth of bacteriae with
mutated DNA/RNA polymerase, preferably wherein the mutation is suitable
to better adapt better to the new substrate.

[0128] Another aspect of the invention relates to a pharmaceutical or
non-pharmaceutical composition comprising the phosphate-modified
nucleosides of the invention being represented by any one of the
structural formulae (I), (I'), (A) and (A'), including any one of the
above-referred specific embodiments thereof. In a particular embodiment,
said non-pharmaceutical composition further comprises one or more natural
NTPs or dNTPs (e.g. ATP,CTP,GTP,UTP,TTP).

[0129] Yet another aspect of the invention relates to a method for the
production of oligonucleotides, RNA, DNA, peptides and/or proteins by
using the phosphate-modified nucleotides of the invention being
represented by any one of the structural formulae (I), (I'), (A) and
(A'), including any one of the above-referred specific embodiments
thereof.

[0130] Another aspect of the present invention relates to compounds
(phosphate-modified nucleosides) represented by any one of the structural
formulae (I), (I'), (A) and (A'), including any one of the above-referred
specific embodiments thereof, having antiviral activity, more
specifically to these compounds that inhibit the replication of viruses,
even more specifically to these compounds that inhibit the replication of
HIV-1.

[0131] Another aspect of the present invention relates to the compounds
(phosphate-modified nucleosides) being represented by any one of the
structural formulae (I), (I'), (A) and (A'), including any one of the
above-referred specific embodiments thereof, for use as a medicine to
treat or prevent a viral infection in a mammal, even more particularly to
treat or prevent a HIV infection in a mammal such as a human being.

[0132] Another aspect of the invention relates to pharmaceutical
compositions comprising the compounds (phosphate-modified nucleosides)
being represented by any one of the structural formulae (I), (I'), (A)
and (A'), including any one of the above-referred specific embodiments
thereof, in combination with one or more pharmaceutical excipients. The
invention further relates to the incorporation of a compound
(phosphate-modified nucleoside) being represented by any one of the
structural formula (I), (I'), (A) or (A'), including any one of the
above-referred specific embodiments thereof, in the manufacture of a
medicament useful for the treatment or prevention of viral infections,
specifically for the treatment of a retroviral infection, and more
specifically for the treatment of a HIV-1 infection. The present
invention also relates to a method of treatment or prevention of a viral
infection, such as a HIV-1 infection, in a mammal such as a human being
by administering to said mammal a therapeutically effective amount, for
instance an antiviral amount, specifically an anti-retroviral amount,
more specifically an anti-HIV-1 amount, of a compound (phosphate-modified
nucleoside) being represented by any one of the structural formula (I),
(I'), (A) or (A'), including any one of the above-referred specific
embodiments thereof.

[0133] Still another aspect of the invention relates to a process for the
preparation of the phosphate-modified nucleosides of the invention. In
one embodiment of said aspect, the method comprises the steps of
interacting a nucleotide monophosphate (NMP) with the methyl ester of an
amino acid to produce the methyl ester of an amino acid phosphoramidate
nucleotide analogue. Deprotection of this nucleoside analogue with an
effective amount of a deprotecting agent such as, but not limited to, an
alkali hydroxide, e.g. 0.04 M NaOH, provides the desired amino acid
phosphoramidate nucleoside of this invention. In an alternative
embodiment of said aspect, the process for the preparation of the
phosphate-modified nucleosides of the invention comprises the synthetic
step as shown in the following Scheme 1:

##STR00011##

wherein (a) schematically represents the presence in the reaction mixture
of an effective amount of a suitable catalyst for the condensation of the
5'-OH and phosphate acid groups. Suitable such catalysts are well known
in the art and include, but are not limited to, arylsulfonylhalides, e.g.
an optionally substituted phenylsulfonyl chloride.

[0155] The term "pyrimidine and purine bases" as used herein includes, but
is not limited to, adenine, thymine, cytosine, uracyl, guanine and
2,6-diaminopurine and analogues thereof. A purine or pyrimidine base as
used herein includes a purine or pyrimidine base found in naturally
occurring nucleosides as mentioned above. An analogue thereof is a base
which mimics such naturally occurring bases in such a way that their
structures (the kinds of atoms and their arrangement) are similar to the
naturally occurring bases but may either possess additional or lack
certain of the functional properties of the naturally occurring bases.
Such analogues include those derived by replacement of a CH moiety by a
nitrogen atom (e.g. 5-azapyrimidines such as 5-azacytosine) or vice versa
(e.g., 7-deazapurines, such as 7-deazaadenine or 7-deazaguanine) or both
(e.g., 7-deaza, 8-azapurines). By derivatives of such bases or analogues
are meant those bases wherein ring substituents are either incorporated,
removed, or modified by conventional substituents known in the art, e.g.
halogen, hydroxyl, amino, (C1-C6)alkyl and others. Such purine
or pyrimidine bases, and analogues thereof, are well known to those
skilled in the art, e.g. as shown at pages 20-38 of WO 03/093290, the
content of which is incorporated herein by reference.

[0156] In particular purine and pyrimidine analogues B for the purpose of
the present invention may be selected from the group comprising
pyrimidine bases represented by the structural formula (B):

##STR00012##

wherein [0157] R7 is selected from the group consisting of --OH,
--SH, --NH2, --NHCH3 and --NHC2H5; [0158] R8 is
selected from the group consisting of hydrogen, methyl, ethyl, isopropyl,
amino, ethylamino, trifluoromethyl, cyano and halogen; and [0159] X is CH
or N, and purine bases represented by the structural formula (D):

##STR00013##

[0159] wherein: [0160] R9 is selected from the group consisting of
hydrogen, --OH, --SH, --NH2, and --NH-Me; [0161] R10 is
selected from the group consisting of hydrogen, methyl, ethyl, hydroxyl,
amino and halogen; and [0162] X and Y are independently selected from CH
and N.

[0163] Just as a few non-limiting examples of pyrimidine analogues, can be
named substituted uracils with the formula (B) wherein X is CH, R7
is hydroxyl, and R8 is methyl, ethyl, isopropyl, amino, ethylamino,
trifluoromethyl, cyano, fluoro, chloro, bromo or iodo.

[0165] As used herein and unless otherwise stated, the term "cycloalkyl"
refers to a monocyclic saturated hydrocarbon monovalent radical having
from 3 to 6 carbon atoms, such as for instance cyclopropyl, cyclobutyl,
cyclopentyl, cyclohexyl, or even up to 10 carbon atoms, such as for
instance cycloheptyl, cyclooctyl and the like, or a C7-10 polycyclic
saturated hydrocarbon monovalent radical having from 7 to 10 carbon atoms
such as, for instance, norbornyl, fenchyl, trimethyltricycloheptyl or
adamantyl.

[0166] The term "(C1-C6) alkoxy" as used herein refers to
substituents wherein a carbon atom of a (C1-C6) alkyl (such as
defined herein), is attached to an oxygen atom through a single bond such
as, but not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy,
iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy.

[0167] As used herein, and unless otherwise stated, the term "aryl"
designates any mono- or polycyclic aromatic monovalent hydrocarbon
radical having from 6 up to 30 carbon atoms such as but not limited to
phenyl, naphthyl, anthracenyl, phenantracyl, fluoranthenyl, chrysenyl,
pyrenyl, biphenylyl, terphenyl, picenyl, indenyl, biphenyl, indacenyl,
benzocyclobutenyl, benzocyclooctenyl and the like, including fused
benzo-cycloalkyl radicals (the latter being as defined above) such as,
for instance, indanyl, tetrahydronaphtyl, fluorenyl and the like, all of
the said radicals being optionally substituted with one or more
substituents independently selected from the group consisting of halogen,
amino, trifluoromethyl, hydroxyl, sulfhydryl, nitro,
(C1-C6)alkoxy, trifluoromethoxy, cyano and
(CH2)q--COOR (wherein R is hydrogen or (C1-C6) alkyl,
and q is 0, 1 or 2), such as for instance 1,2-dicarboxylphenyl,
1,3-dicarboxylphenyl, 4-fluorophenyl, 4-chlorophenyl, 3,4-dichlorophenyl,
4-cyanophenyl, 2,6-dichlorophenyl, 2-fluorophenyl, 3-chlorophenyl,
3,5-dichlorophenyl and the like.

[0168] As used herein and unless otherwise stated, the term halogen refers
to any atom selected from the group consisting of fluorine (F), chlorine
(Cl), bromine (Br) and iodine (I).

[0169] Any substituent designation that is found in more than one site in
a phosphate-modified nucleotide of this invention shall be independently
selected.

[0171] It will be appreciated by those skilled in the art that
phosphate-modified nucleotides of the invention having a chiral center
may exist in and be isolated in optically active and racemic forms. Some
phosphate-modified nucleotides may exhibit polymorphism. It is to be
understood that the present invention encompasses any racemic,
optically-active, polymorphic, or stereoisomeric form, or mixtures
thereof, of a phosphate-modified nucleotide of the invention, which
possess the useful properties described herein, it being well known in
the art how to prepare optically active forms (for example, by resolution
of the racemic form by recrystallization techniques, by synthesis from
optically-active starting materials, by chiral synthesis, or by
chromatographic separation using a chiral stationary phase).

[0172] The term "salt" as used herein, refers to the ionic product of a
reaction between an acid and a base, and embraces the "pharmaceutically
acceptable salts" as described in details below. Salts of the
phosphate-modified nucleosides represented by any one of the structural
formulae (I), (I'), (A) and (A'), including any one of the particular
embodiments thereof, may be formed at any acid or base functionality
within the compound, in particular, R3, R4 and R5 may
represent or comprise an acid or base functionality. In particular, salts
of the phosphate-modified nucleosides represented by any one of the
structural formulae (I), (I'), (A) and (A') may be formed as follows.
When R3 is hydrogen, it is acidic and may therefore engage in salt
formation with an inorganic or organic base. R4 and R5 may
comprise acidic functionalities such as carboxylic groups (COOH), which
can equally engage in salt formation with an organic or inorganic base.
Alternatively, R4 and R5 may comprise base functionalities,
such as imidazolyl, which in turn can engage in salt formation with an
organic or inorganic acid.

[0173] Certain of the phosphate-modified nucleosides described herein are
capable of acting as prodrugs when substituted with appropriately
selected functional groups. These are labile functional groups which
separate from an active inhibitory phosphate-modified nucleoside during
metabolism, systemically, inside a cell, by hydrolysis, enzymatic
cleavage, or by some other process (see e.g. Bundgaard et al., Design and
Application of Prodrugs in "Textbook of Drug Design and Development"
(1991), Eds. Harwood Academic Publishers, pp. 113-191), the content of
which is incorporated herein by reference. These prodrug moieties can
serve to enhance solubility, absorption and lipophilicity to optimize
drug delivery, bioavailability and efficacy. A "prodrug" may thus be a
covalently modified analogue of a therapeutically active
phosphate-modified nucleoside as defined herein, and can be
therapeutically active in its own right.

[0174] The term "prodrug", as used herein, more specifically relates to an
inactive or significantly less active derivative of a phosphate-modified
nucleoside represented by any one of the structural formulae (I), (I'),
(A) and (A'), including any one of the particular embodiments thereof,
which undergoes spontaneous or enzymatic transformation within the body
of a mammal (e.g. a human being) in order to release the
pharmacologically active form of the compound. For a comprehensive
review, see Rautio J. et al. in "Prodrug design and clinical
applications, Nature Reviews Drug Discovery" (2008) doi:
10.1038/nrd2468), the content of which is incorporated herein by
reference. In particular for the purpose of the present invention,
prodrugs of the phosphate-modified nucleosides represented by any one of
the structural formulae (I), (I'), (A) and (A'), including any one of the
particular embodiments thereof, may be formed as follows. When R3 is
hydrogen, a free phosphate acid function is available for prodrug
formation as described in detail by Hecker S. J. et al in "Prodrugs of
phosphates and phosphonates, Journal of Medicinal Chemistry" (2008) doi:
10.1021/jm701260b). R4 and R5 comprise acid functionalities
such as carboxylic acid groups (COOH) which may be used for the formation
of a prodrug. Such carboxylic acid prodrug may occur in the form of an
ester, in particular acyloxyalkylesters such as, but not limited to, a
pivaloyloxymethyl ester (POM), an isopropyloxy-carbonyloxymethyl ester
(POC) or a S-acyl-2-thioethyl (SATE) ester, a carbonate, a carbamate, or
an amide, such as may be derived from amino acids.

[0175] The term "peptide" as used herein refers to a sequence of 2 to 50
amino acids (e.g. as defined hereinabove) or peptidyl residues. The
sequence may be linear or cyclic. Preferably a peptide comprises 2 to 25,
or 5 to 20 amino acids.

[0176] The term "oligonucleotide" as used herein refers to a
polynucleotide formed by a plurality of linked nucleotide units. The
nucleotide units each include a nucleoside unit linked together via a
phosphate linking group. These nucleotides can be natural or modified in
their phosphate, sugar or nucleobase group. The oligonucleotide may be
naturally occurring or non naturally occurring.

[0177] The term "polymerase" as used herein refers to an enzyme that can
synthesize DNA or RNA from a DNA or RNA template and includes but is not
limited to Therminator DNA polymerase, KF(exo.sup.-)DNA polymerase and
HIV Reverse Transcriptase.

Biological Applications of the Invention

[0178] Amino acid phosphoramidates, phospho-esters and phospho-thioesters
according to this invention may be used as alternative substrates and
biotechnology tools.

[0179] Fast emerging applications of modified nucleosides as biotechnology
tools also require new and efficient ways to synthesize DNA and RNA
building blocks such as nucleoside triphosphates and amidites for the
use, for example, in PCR, labeling, or enzymatic incorporation of
nucleotides, and in the automated DNA synthesis, respectively.
Furthermore, some biotechnology applications require incorporation of a
nucleotide by enzymatic means using DNA or RNA polymerases. However, at
times, due to chemical nature and modifications present in the modified
nucleosides, triphosphate synthesis is not always feasible and/or
provides insufficient and low yields.

[0180] Thus, the invention relates to a method for preparing an
oligonucleotide comprising phosphate-modified nucleosides, comprising the
step of incorporating at least one of said phosphate-modified nucleosides
into a DNA/RNA strand, wherein said phosphate-modified nucleoside is
represented by any one of the structural formulae (A), (I), (A') and
(I'), including any one of the specific embodiments described herein.
Within the general framework of such a method, one or more of the
following embodiments may be particularly useful: [0181] the
oligonucleotide may be prepared by DNA/RNA polymerase-dependent
amplification; [0182] said DNA/RNA polymerase-dependent amplification is
PCR; [0183] the oligonucleotide may be prepared by administering said
phosphate-modified nucleosides to bacteriae comprising a DNA/RNA
polymerase; [0184] said polymerase may be from a microorganism or from
bacterial or viral origin; [0185] said polymerase may be Therminator DNA
polymerase; KF (exo.sup.-) DNA polymerase or Reverse Transcriptase.

[0186] Therefore, an amino acid (such as, but not limited to, aspartic
acid) coupled to a nucleoside monophosphate through a phosphoramidate
(P--N) bond can serve as an alternative or substitute group to a
pyrophosphate moiety. However, fitting into an active site and the
subsequent nucleotidyl transfer may be less efficient for an amino acid
phosphoramidate (e.g. Asp-dAMP) compared to the natural substrates/dNTPs
(e.g. dATP) for the natural polymerase/enzyme. In this situation, mutated
polymerases can be used to increase the efficiency of recognition and
incorporation of amino acid phosphoramidate nucleotides. Such an
embodiment of the invention with mutated polymerases can be used to
specifically select or grow bacteriae by using these amino acid
phosphoramidate nucleosides as a substrate. An additional advantage of
this application is that polymerases that demonstrated efficient
recognition and incorporation of amino acid phosphoramidate nucleosides
in our studies are also shown to tolerate various sugar modifications and
unnatural nucleobases quite well. Therefore, the enzymatic synthesis of
DNA and, RNA sequences containing unnatural nucleobases can be
accomplished whilst avoiding at times cumbersome nucleoside triphosphate
synthesis and purification.

[0187] The amino acid phosphoramidates, phospho-esters and
phospho-thioesters of this invention are also useful as antiviral
compounds

[0188] The compounds of the invention can be employed for the treatment of
viral infections, more particularly Human Immunodeficiency Virus (HIV)
infections, in particular of Human Immunodeficiency Virus type 1 (HIV-1).
When using one or more derivatives represented by any one of the
structural formulae (I), (I'), (A) and (A'), including any one of the
particular embodiments thereof, as defined herein: [0189] the active
ingredients of the compound(s) may be administered to the mammal
(including a human being) to be treated by any means well known in the
art, i.e. orally, intranasally, subcutaneously, intramuscularly,
intradermally, intravenously, intra-arterially, parenterally or by
catheterization. [0190] the therapeutically effective amount of the
preparation of the compound(s), especially for the treatment of viral
infections in humans and other mammals, preferably is a HIV enzyme
inhibiting amount. More preferably, it is a HIV replication inhibiting
amount or a HIV enzyme (in particular reverse transcriptase) inhibiting
amount of the derivative(s) represented by any one of the structural
formulae (I), (I'), (A) and (A'), including any one of the particular
embodiments thereof, corresponds to an amount which ensures a plasma
level of between 1 μg/ml and 100 mg/ml, optionally of 10 mg/ml.
Depending upon the pathologic condition to be treated and the patient's
condition, the said effective amount may be divided into several
sub-units per day or may be administered at more than one day intervals.

[0191] The present invention further relates to a method for preventing or
treating a viral infection in a subject or patient by administering to
the patient in need thereof a therapeutically effective amount of the
compounds of the present invention. The therapeutically effective amount
of the compound(s), especially for the treatment of viral infections in
humans and other mammals, preferably is a HIV enzyme inhibiting amount.
More preferably, it is a HIV replication inhibiting amount or a HIV
enzyme (in particular reverse transcriptase) inhibiting amount of the
derivative(s) represented by any one of the structural formulae (I),
(I'), (A) and (A'), including any one of the particular embodiments
thereof. Depending upon the pathologic condition to be treated and the
patient's condition, the said effective amount may be divided into
several sub-units per day or may be administered at more than one-day
intervals.

[0192] The present invention also relates to a combination of different
antiviral drugs of the invention or to a combination of the antiviral
drugs of the invention with one or more other drugs that exhibit anti-HIV
activity.

[0193] The invention also relates to a combined preparation of antiviral
drugs which may be either:

A) a composition comprising [0194] (a) a combination of two or more of
the compounds of the present invention, and [0195] (b) optionally one or
more pharmaceutical excipients or pharmaceutically acceptable carriers,
for simultaneous, separate or sequential use in the treatment or
prevention of a viral infection, or B) a composition comprising [0196]
(c) one or more anti-viral agents, and [0197] (d) at least one compound
of the present invention, and [0198] (e) optionally one or more
pharmaceutical excipients or pharmaceutically acceptable carriers, for
simultaneous, separate or sequential use in the treatment or prevention
of a viral infection.

[0199] Suitable anti-viral agents (c) for inclusion into the antiviral
combined preparations of this invention include for instance, inhibitors
of BVDV or HCV replication respectively, such as interferon-alfa (either
pegylated or not), ribavirin and other selective inhibitors of the
replication of HCV, such as a compound disclosed in EP1162196, WO
03/010141, WO 03/007945, WO 03/010140 and WO 00/204425 and/or an
inhibitor of flaviviral protease and/or one or more additional flavivirus
polymerase inhibitors.

[0200] The pharmaceutical composition or combined preparation with
activity against viral infection according to this invention may contain
a compound of the present invention over a broad content range depending
on the contemplated use and the expected effect of the preparation.
Generally, the content of the compound of the present invention in the
combined preparation is within the range of 0.1 to 99.9% by weight,
preferably from 1 to 99% by weight, more preferably from 5 to 95% by
weight.

[0201] When using a pharmaceutical composition of combined preparation:
[0202] the active ingredients may be administered to the mammal
(including a human) to be treated by any means well known in the art,
i.e. orally, intranasally, subcutaneously, intramuscularly,
intradermally, intravenously, intra-arterially, parenterally or by
catheterization; and/or [0203] the therapeutically effective amount of
each of the active agents, especially for the treatment of viral
infections in humans and other mammals, particularly is a HIV enzyme
inhibiting amount.

[0204] When applying a combined preparation, the active ingredients may be
administered simultaneously but it is also beneficial to administer them
separately or sequentially, for instance within a relatively short period
of time (e.g. within about 24 hours) in order to achieve their functional
fusion in the body to be treated.

[0205] The invention also relates to the compounds of the formulae
described herein being used for inhibition of the proliferation of other
viruses than HIV-1, particularly for the inhibition of other members of
the family of the retroviruses.

[0206] The present invention further provides veterinary compositions
comprising at least one active ingredient as above defined together with
a veterinary carrier therefor. Veterinary carriers are materials useful
for the purpose of administering the composition and may be solid, liquid
or gaseous materials which are otherwise inert or acceptable in the
veterinary art and are compatible with the active ingredient. These
veterinary compositions may be administered orally, parenterally or by
any other desired route.

[0207] More generally, the invention relates to the compounds represented
by any one of the structural formulae (I), (I'), (A) and (A'), including
any one of the particular embodiments thereof, being useful as agents
having biological activity (particularly antiviral activity) or as
diagnostic agents.

[0208] The compounds of the invention optionally are bound covalently to
an insoluble matrix and used for affinity chromatography separations,
depending on the nature of the groups of the compounds, for example
compounds with pendant aryl are useful in hydrophobic affinity
separations.

[0209] Those of skill in the art will also recognize that the compounds of
the invention may exist in many different protonation states, depending
on, among other things, the pH of their environment. While the structural
formulae provided herein depict the compounds in only one of several
possible protonation states, it will be understood that these structures
are illustrative only, and that the invention is not limited to any
particular protonation state--any and all protonated forms of the
compounds are intended to fall within the scope of the invention.

[0210] The term "pharmaceutically acceptable salts" as used herein means
the therapeutically active non-toxic salt forms which the compounds
(phosphate-modified nucleosides) represented by any one of the structural
formulae (I), (I'), (A) and (A'), including any one of the particular
embodiments thereof, are able to form. Therefore, the compounds of this
invention optionally comprise salts of the compounds herein, especially
pharmaceutically acceptable non-toxic salts containing, for example,
Na.sup.+, Li.sup.+, K.sup.+, Ca2+ and Mg2+. Such salts may
include those derived by combination of appropriate cations such as
alkali and alkaline earth metal ions or ammonium and quaternary amino
ions with an acid anion moiety, typically a carboxylic acid. The
compounds of the invention may bear multiple positive or negative
charges. The net charge of the compounds of the invention may be either
positive or negative. Any associated counter ions are typically dictated
by the synthesis and/or isolation methods by which the compounds are
obtained. Typical counter ions include, but are not limited to ammonium,
sodium, potassium, lithium, halides, acetate, trifluoroacetate, etc., and
mixtures thereof. It will be understood that the identity of any
associated counter ion is not a critical feature of the invention, and
that the invention encompasses the compounds in association with any type
of counter ion. Moreover, as the compounds can exist in a variety of
different forms, the invention is intended to encompass not only forms of
the compounds that are in association with counter ions (e.g., dry
salts), but also forms that are not in association with counter ions
(e.g., aqueous or organic solutions). Metal salts typically are prepared
by reacting the metal hydroxide with a compound of this invention.
Examples of metal salts which are prepared in this way are salts
containing Li.sup.+, Na.sup.+, and K.sup.+. A less soluble metal salt can
be precipitated from the solution of a more soluble salt by addition of
the suitable metal compound. In addition, salts may be formed from acid
addition of certain organic and inorganic acids to basic centers,
typically amines, or to acidic groups. Examples of such appropriate acids
include, for instance, inorganic acids such as hydrohalic acids, e.g.
hydrochloric or hydrobromic acid, sulfuric acid, nitric acid, phosphoric
acid and the like; or organic acids such as, for example, acetic,
propanoic, hydroxyacetic, 2-hydroxypropanoic, 2-oxopropanoic, lactic,
pyruvic, oxalic (i.e. ethanedioic), malonic, succinic (i.e. butanedioic
acid), maleic, fumaric, malic, tartaric, citric, methanesulfonic,
ethanesulfonic, benzene-sulfonic, p-toluenesulfonic, cyclohexanesulfamic,
salicylic (i.e. 2-hydroxybenzoic), p-aminosalicylic and the like.
Furthermore, this term also includes the solvates which the compounds
represented by any one of the structural formulae (I), (I'), (A) and
(A'), including any one of the particular embodiments thereof, as well as
their salts are able to form, such as for example hydrates, alcoholates,
nitriles and the like. Finally, it is to be understood that the
compositions herein may comprise compounds of the invention in their
unionized, as well as zwitterionic form, and combinations with
stoichiometric amounts of water as in hydrates.

[0211] Also included within the scope of this invention are the salts of
the parental compounds with one or more amino acids, especially the
naturally-occurring amino acids found as protein components, as well as
known non-natural analogues thereof. The amino acid typically is one
bearing a side chain with a basic or acidic group, e.g., lysine, arginine
or glutamic acid, or a neutral group such as glycine, serine, threonine,
alanine, isoleucine, or leucine.

[0212] The compounds (phosphate-modified nucleosides) of the invention
also include physiologically acceptable salts thereof. Examples of
physiologically acceptable salts of the compounds (phosphate-modified
nucleosides) of the invention include salts derived from an appropriate
base, such as an alkali metal (for example, sodium), an alkaline earth
(for example, magnesium), ammonium and NA4.sup.+ (wherein A is
C1-C4 alkyl). Physiologically acceptable salts of an hydrogen
atom or an amino group include salts of organic carboxylic acids such as
acetic, benzoic, lactic, fumaric, tartaric, maleic, malonic, malic,
isethionic, lactobionic and succinic acids; organic sulfonic acids, such
as methanesulfonic, ethanesulfonic, benzenesulfonic and p-toluenesulfonic
acids; and inorganic acids, such as hydrochloric, sulfuric, phosphoric
and sulfamic acids. Physiologically acceptable salts of a compound
containing a hydroxy group include the anion of said compound in
combination with a suitable cation such as Na.sup.+ and NA4.sup.+
(wherein A typically is independently selected from H or a
C1-C4 alkyl group). However, salts of acids or bases which are
not physiologically acceptable may also find use, for example, in the
preparation or purification of a physiologically acceptable compound. All
salts, whether or not derived form a physiologically acceptable acid or
base, are within the scope of the present invention.

[0213] As used herein and unless otherwise stated, the term "enantiomer"
means each individual optically active form of a compound of the
invention, having an optical purity or enantiomeric excess (as determined
by methods standard in the art) of at least 80% (i.e. at least 90% of one
enantiomer and at most 10% of the other enantiomer), preferably at least
90% and more preferably at least 98%.

[0214] The term "isomers" as used herein means all possible isomeric
forms, including tautomeric and stereochemical forms, which the compounds
of formula I may possess, but not including position isomers. Typically,
the structures shown herein exemplify only one tautomeric or resonance
form of the compounds, but the corresponding alternative configurations
are contemplated as well. Unless otherwise stated, the chemical
designation of compounds denotes the mixture of all possible
stereochemically isomeric forms, said mixtures containing all
diastereomers and enantiomers (since the compounds of formula I may have
at least one chiral center) of the basic molecular structure, as well as
the stereochemically pure or enriched compounds. More particularly,
stereogenic centers may have either the R- or S-configuration, and
multiple bonds may have either cis- or trans-configuration. Pure isomeric
forms of the said compounds are defined as isomers substantially free of
other enantiomeric or diastereomeric forms of the same basic molecular
structure. In particular, the term "stereoisomerically pure" or "chirally
pure" relates to compounds having a stereoisomeric excess of at least
about 80% (i.e. at least 90% of one isomer and at most 10% of the other
possible isomers), preferably at least 90%, more preferably at least 94%
and most preferably at least 97%. The terms "enantiomerically pure" and
"diastereomerically pure" should be understood in a similar way, having
regard to the enantiomeric excess, respectively the diastereomeric
excess, of the mixture in question. Separation of stereoisomers is
accomplished by standard methods known to those in the art. One
enantiomer of a compound of the invention can be separated substantially
free of its opposing enantiomer by a method such as formation of
diastereomers using optically active resolving agents (see e.g.
"Stereochemistry of Carbon Compounds" (1962) by E. L. Eliel, McGraw Hill;
and Lochmuller in J. Chromatogr. (1975) 113:(3) 283-302). Separation of
isomers in a mixture can be accomplished by any suitable method,
including: (1) formation of ionic, diastereomeric salts with chiral
compounds and separation by fractional crystallization or other methods,
(2) formation of diastereomeric compounds with chiral derivatizing
reagents, separation of the diastereomers, and conversion to the pure
enantiomers, or (3) enantiomers can be separated directly under chiral
conditions. Under method (1), diastereomeric salts can be formed by
reaction of enantiomerically pure chiral bases such as brucine, quinine,
ephedrine, strychnine, a-methyl-b-phenylethylamine (amphetamine), and the
like with asymmetric compounds bearing acidic functionality, such as
carboxylic acid and sulfonic acid. The diastereomeric salts may be
induced to separate by fractional crystallization or ionic
chromatography. For separation of the optical isomers of amino compounds,
addition of chiral carboxylic or sulfonic acids, such as camphorsulfonic
acid, tartaric acid, mandelic acid, or lactic acid can result in
formation of the diastereomeric salts. Alternatively, by method (2), the
substrate to be resolved may be reacted with one enantiomer of a chiral
compound to form a diastereomeric pair (Eliel et al in Stereochemistry of
Organic Compounds (1994) John Wiley & Sons, Inc., p. 322). Diastereomeric
compounds can be formed by reacting asymmetric compounds with
enantiomerically pure chiral derivatizing reagents, such as menthyl
derivatives, followed by separation of the diastereomers and hydrolysis
to yield the free, enantiomerically enriched xanthene. A method of
determining optical purity involves making chiral esters, such as a
menthyl ester or Mosher ester, a-methoxy-a-(trifluoromethyl)phenyl
acetate (Jacob III. (1982) J. Org. Chem. 47:4165), of the racemic
mixture, and analyzing the NMR spectrum for the presence of the two
atropisomeric diastereomers. Stable diastereomers can be separated and
isolated by normal- and reverse-phase chromatography following methods
for separation of atropisomeric naphthyl-isoquinolines (Hoye, T.,
WO96/15111). Under method (3), a racemic mixture of two asymmetric
enantiomers is separated by chromatography using a chiral stationary
phase. Suitable chiral stationary phases are, for example,
polysaccharides, in particular cellulose or amylose derivatives.
Commercially available polysaccharide based chiral stationary phases are
ChiralCel® CA, OA, OB5, OC5, OD, OF, OG, OJ and OK, and Chiralpak®
AD, AS, OP(+) and OT(+). Appropriate eluents or mobile phases for use in
combination with said polysaccharide chiral stationary phases are hexane
and the like, modified with an alcohol such as ethanol, isopropanol and
the like.

[0215] The terms cis and trans are used herein in accordance with Chemical
Abstracts nomenclature and include reference to the position of the
substituents on a ring moiety. The absolute stereochemical configuration
of the compounds of formula I may easily be determined by those skilled
in the art while using well-known methods such as, for example, X-ray
diffraction.

[0216] The compounds of the invention may be formulated with conventional
carriers and excipients, which will be selected in accord with ordinary
practice. Tablets will contain excipients, glidants, fillers, binders and
the like. Aqueous formulations are prepared in sterile form, and when
intended for delivery by other than oral administration generally will be
isotonic. Formulations optionally contain excipients such as those set
forth in the "Handbook of Pharmaceutical Excipients" (1986) and include
ascorbic acid and other antioxidants, chelating agents such as EDTA,
carbohydrates such as dextrin, hydroxyalkylcellulose,
hydroxyalkylmethylcellulose, stearic acid and the like.

[0217] Subsequently, the term "pharmaceutically acceptable carrier" as
used herein means any material or substance with which the active
ingredient is formulated in order to facilitate its application or
dissemination to the locus to be treated, for instance by dissolving,
dispersing or diffusing the said composition, and/or to facilitate its
storage, transport or handling without impairing its effectiveness. The
pharmaceutically acceptable carrier may be a solid or a liquid or a gas
which has been compressed to form a liquid, i.e. the compositions of this
invention can suitably be used as concentrates, emulsions, solutions,
granulates, dusts, sprays, aerosols, suspensions, ointments, creams,
tablets, pellets or powders.

[0218] Suitable pharmaceutical carriers for use in the said pharmaceutical
compositions and their formulation are well known to those skilled in the
art, and there is no particular restriction to their selection within the
present invention. They may also include additives such as wetting
agents, dispersing agents, stickers, adhesives, emulsifying agents,
solvents, coatings, antibacterial and antifungal agents (for example
phenol, sorbic acid, chlorobutanol), isotonic agents (such as sugars or
sodium chloride) and the like, provided the same are consistent with
pharmaceutical practice, i.e. carriers and additives which do not create
permanent damage to mammals. The pharmaceutical compositions of the
present invention may be prepared in any known manner, for instance by
homogeneously mixing, coating and/or grinding the active ingredients, in
a one-step or multi-steps procedure, with the selected carrier material
and, where appropriate, the other additives such as surface-active
agents. They may also be prepared by micronisation, for instance in view
to obtain them in the form of microspheres usually having a diameter of
about 1 to 10 μm, namely for the manufacture of microcapsules for
controlled or sustained release of the active ingredients.

[0219] Suitable surface-active agents, also known as emulgent or
emulsifier, to be used in the pharmaceutical compositions of the present
invention are non-ionic, cationic and/or anionic materials having good
emulsifying, dispersing and/or wetting properties. Suitable anionic
surfactants include both water-soluble soaps and water-soluble synthetic
surface-active agents. Suitable soaps are alkaline or alkaline-earth
metal salts, unsubstituted or substituted ammonium salts of higher fatty
acids (C10-C22), e.g. the sodium or potassium salts of oleic or
stearic acid, or of natural fatty acid mixtures obtainable form coconut
oil or tallow oil. Synthetic surfactants include sodium or calcium salts
of polyacrylic acids; fatty sulphonates and sulphates; sulphonated
benzimidazole derivatives and alkylarylsulphonates. Fatty sulphonates or
sulphates are usually in the form of alkaline or alkaline-earth metal
salts, unsubstituted ammonium salts or ammonium salts substituted with an
alkyl or acyl radical having from 8 to 22 carbon atoms, e.g. the sodium
or calcium salt of lignosulphonic acid or dodecylsulphonic acid or a
mixture of fatty alcohol sulphates obtained from natural fatty acids,
alkaline or alkaline-earth metal salts of sulphuric or sulphonic acid
esters (such as sodium lauryl sulphate) and sulphonic acids of fatty
alcohol/ethylene oxide adducts. Suitable sulphonated benzimidazole
derivatives preferably contain 8 to 22 carbon atoms. Examples of
alkylarylsulphonates are the sodium, calcium or alcanolamine salts of
dodecylbenzene sulphonic acid or dibutyl-naphtalenesulphonic acid or a
naphtalene-sulphonic acid/formaldehyde condensation product. Also
suitable are the corresponding phosphates, e.g. salts of phosphoric acid
ester and an adduct of p-nonylphenol with ethylene and/or propylene
oxide, or phospholipids. Suitable phospholipids for this purpose are the
natural (originating from animal or plant cells) or synthetic
phospholipids of the cephalin or lecithin type such as e.g.
phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerine,
lysolecithin, cardiolipin, dioctanyiphosphatidyl-choline,
dipalmitoylphosphatidyl-choline and their mixtures.

[0222] A more detailed description of surface-active agents suitable for
this purpose may be found for instance in "McCutcheon's Detergents and
Emulsifiers Annual" (MC Publishing Crop., Ridgewood, N.J., 1981),
"Tensid-Taschenbucw", 2 d ed. (Hanser Verlag, Vienna, 1981) and
"Encyclopaedia of Surfactants" (Chemical Publishing Co., New York, 1981),
the contents of which is incorporated herein by reference.

[0223] Compounds of the invention and their physiologically acceptable
salts (hereafter collectively referred to as the active ingredients) may
be administered by any route appropriate to the condition to be treated,
suitable routes including oral, rectal, nasal, topical (including ocular,
buccal and sublingual), vaginal and parenteral (including subcutaneous,
intramuscular, intravenous, intradermal, intrathecal and epidural). The
preferred route of administration may vary with for example the condition
of the recipient.

[0224] While it is possible for the active ingredients to be administered
alone it is preferable to present them as pharmaceutical formulations.
The formulations, both for veterinary and for human use, of the present
invention comprise at least one active ingredient, as above described,
together with one or more pharmaceutically acceptable carriers therefore
and optionally other therapeutic ingredients. The carrier(s) optimally
are "acceptable" in the sense of being compatible with the other
ingredients of the formulation and not deleterious to the recipient
thereof. The formulations include those suitable for oral, rectal, nasal,
topical (including buccal and sublingual), vaginal or parenteral
(including subcutaneous, intramuscular, intravenous, intradermal,
intrathecal and epidural) administration. The formulations may
conveniently be presented in unit dosage form and may be prepared by any
of the methods well known in the art of pharmacy. Such methods include
the step of bringing into association the active ingredient with the
carrier which constitutes one or more accessory ingredients. In general
the formulations are prepared by uniformly and intimately bringing into
association the active ingredient with liquid carriers or finely divided
solid carriers or both, and then, if necessary, shaping the product.

[0225] Formulations of the present invention suitable for oral
administration may be presented as discrete units such as capsules,
cachets or tablets each containing a predetermined amount of the active
ingredient; as a powder or granules; as solution or a suspension in an
aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid
emulsion or a water-in-oil liquid emulsion. The active ingredient may
also be presented as a bolus, electuary or paste.

[0226] A tablet may be made by compression or molding, optionally with one
or more accessory ingredients. Compressed tablets may be prepared by
compressing in a suitable machine the active ingredient in a free-flowing
form such as a powder or granules, optionally mixed with a binder,
lubricant, inert diluent, preservative, surface active or dispersing
agent. Molded tablets may be made by molding in a suitable machine a
mixture of the powdered compound moistened with an inert liquid diluent.
The tablets may optionally be coated or scored and may be formulated so
as to provide slow or controlled release of the active ingredient
therein. For infections of the eye or other external tissues e.g. mouth
and skin, the formulations are optionally applied as a topical ointment
or cream containing the active ingredient(s) in an amount of, for
example, 0.075 to 20% w/w (including active ingredient(s) in a range
between 0.1% and 20% in increments of 0.1% w/w such as 0.6% w/w, 0.7%
w/w, etc), preferably 0.2 to 15% w/w and most preferably 0.5 to 10% w/w.
When formulated in an ointment, the active ingredients may be employed
with either a paraffinic or a water-miscible ointment base.
Alternatively, the active ingredients may be formulated in a cream with
an oil-in-water cream base. If desired, the aqueous phase of the cream
base may include, for example, at least 30% w/w of a polyhydric alcohol,
i.e. an alcohol having two or more hydroxyl groups such as propylene
glycol, butane 1,3-diol, mannitol, sorbitol, glycerol and polyethylene
glycol (including PEG400) and mixtures thereof. The topical formulations
may desirably include a compound which enhances absorption or penetration
of the active ingredient through the skin or other affected areas.
Examples of such dermal penetration enhancers include dimethylsulfoxide
and related analogs.

[0227] The oily phase of the emulsions of this invention may be
constituted from known ingredients in a known manner. While the phase may
comprise merely an emulsifier (otherwise known as an emulgent), it
desirably comprises a mixture of at least one emulsifier with a fat or an
oil or with both a fat and an oil. Optionally, a hydrophilic emulsifier
is included together with a lipophilic emulsifier which acts as a
stabilizer. It is also preferred to include both an oil and a fat.
Together, the emulsifier(s) with or without stabilizer(s) make up the
so-called emulsifying wax, and the wax together with the oil and fat make
up the so-called emulsifying ointment base which forms the oily dispersed
phase of the cream formulations.

[0228] The choice of suitable oils or fats for the formulation is based on
achieving the desired cosmetic properties, since the solubility of the
active compound in most oils likely to be used in pharmaceutical emulsion
formulations is very low. Thus the cream should optionally be a
non-greasy, non-staining and washable product with suitable consistency
to avoid leakage from tubes or other containers. Straight or branched
chain, mono- or dibasic alkyl esters such as di-isoadipate, isocetyl
stearate, propylene glycol diester of coconut fatty acids, isopropyl
myristate, decyl oleate, isopropyl palmitate, butyl stearate,
2-ethylhexyl palmitate or a blend of branched chain esters known as
Crodamol CAP may be used, the last three being preferred esters. These
may be used alone or in combination depending on the properties required.
Alternatively, high melting point lipids such as white soft paraffin
and/or liquid paraffin or other mineral oils can be used.

[0229] Formulations suitable for topical administration to the eye also
include eye drops wherein the active ingredient is dissolved or suspended
in a suitable carrier, especially an aqueous solvent for the active
ingredient. The active ingredient is optionally present in such
formulations in a concentration of 0.5 to 20%, advantageously 0.5 to 10%
particularly about 1.5% w/w. Formulations suitable for topical
administration in the mouth include lozenges comprising the active
ingredient in a flavored basis, usually sucrose and acacia or tragacanth;
pastilles comprising the active ingredient in an inert basis such as
gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising
the active ingredient in a suitable liquid carrier.

[0230] Formulations for rectal administration may be presented as a
suppository with a suitable base comprising for example cocoa butter or a
salicylate. Formulations suitable for nasal administration wherein the
carrier is a solid include a coarse powder having a particle size for
example in the range 20 to 500 microns (including particle sizes in a
range between 20 and 500 microns in increments of 5 microns such as 30
microns, 35 microns, etc), which is administered in the manner in which
snuff is taken, i.e. by rapid inhalation through the nasal passage from a
container of the powder held close up to the nose. Suitable formulations
wherein the carrier is a liquid, for administration as for example a
nasal spray or as nasal drops, include aqueous or oily solutions of the
active ingredient. Formulations suitable for aerosol administration may
be prepared according to conventional methods and may be delivered with
other therapeutic agents.

[0231] Formulations suitable for vaginal administration may be presented
as pessaries, tampons, creams, gels, pastes, foams or spray formulations
containing in addition to the active ingredient such carriers as are
known in the art to be appropriate.

[0232] Formulations suitable for parenteral administration include aqueous
and non-aqueous sterile injection solutions which may contain
anti-oxidants, buffers, bacteriostats and solutes which render the
formulation isotonic with the blood of the intended recipient; and
aqueous and non-aqueous sterile suspensions which may include suspending
agents and thickening agents. The formulations may be presented in
unit-dose or multi-dose containers, for example sealed ampoules and
vials, and may be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid carrier, for example
water for injections, immediately prior to use. Extemporaneous injection
solutions and suspensions may be prepared from sterile powders, granules
and tablets of the kind previously described.

[0233] Preferred unit dosage formulations are those containing a daily
dose or unit daily sub-dose, as herein above recited, or an appropriate
fraction thereof, of an active ingredient.

[0234] It should be understood that in addition to the ingredients
particularly mentioned above the formulations of this invention may
include other agents conventional in the art having regard to the type of
formulation in question, for example those suitable for oral
administration may include flavoring agents.

[0235] This invention includes controlled release pharmaceutical
formulations containing as active ingredient one or more compounds of the
invention ("controlled release formulations") in which the release of the
active ingredient can be controlled and regulated to allow less frequency
dosing or to improve the pharmacokinetic or toxicity profile of a given
invention compound. Controlled release formulations adapted for oral
administration in which discrete units comprising one or more compounds
of the invention can be prepared according to conventional methods.

[0236] Additional ingredients may be included in order to control the
duration of action of the active ingredient in the composition. Control
release compositions may thus be achieved by selecting appropriate
polymer carriers such as for example polyesters, polyamino acids,
polyvinyl pyrrolidone, ethylene-vinyl acetate copolymers,
methylcellulose, carboxymethylcellulose, protamine sulfate and the like.
The rate of drug release and duration of action may also be controlled by
incorporating the active ingredient into particles, e.g. microcapsules,
of a polymeric substance such as hydrogels, polylactic acid,
hydroxymethylcellulose, polymethyl methacrylate and the other
above-described polymers. Such methods include colloid drug delivery
systems like liposomes, microspheres, microemulsions, nanoparticles,
nanocapsules and so on. Depending on the route of administration, the
pharmaceutical composition may require protective coatings.
Pharmaceutical forms suitable for injectionable use include sterile
aqueous solutions or dispersions and sterile powders for the
extemporaneous preparation thereof. Typical carriers for this purpose
therefore include biocompatible aqueous buffers, ethanol, glycerol,
propylene glycol, polyethylene glycol and the like and mixtures thereof.

[0237] In view of the fact that, when several active ingredients are used
in combination, they do not necessarily bring out their joint therapeutic
effect directly at the same time in the mammal to be treated, the
corresponding composition may also be in the form of a medical kit or
package containing the two ingredients in separate but adjacent
repositories or compartments. In the latter context, each active
ingredient may therefore be formulated in a way suitable for an
administration route different from that of the other ingredient, e.g.
one of them may be in the form of an oral or parenteral formulation
whereas the other is in the form of an ampoule for intravenous injection
or an aerosol.

[0238] The presented invention shows that a phosphate-modified nucleoside
represented by any one of the structural formulae (I), (I'), (A) and
(A'), including any one of the particular embodiments thereof, such as
but not limited to 2'-deoxy-adenine-5'-aspartyl-phosphoramidate is
successfully recognized and efficiently incorporated into a growing DNA
strand by HIV RT and Therminator DNA polymerase. This means that an
aspartyl-phosphoramidate moiety can mimic a pyrophosphate group and
behave as a good leaving group in a nucleotidyl transfer. Incorporation
of phosphoramidate analogues, although to a lesser extent, was also
observed for histidyl and glycinyl phosphoramidates, respectively.
Therefore, it is feasible to use chain-terminating nucleotides coupled to
aspartic acid through a phosphoramidate linkage for a direct inhibition
of HIV RT or other viral polymerases as depicted in FIG. 2A. Effective
inhibition of HIV RT or other viral polymerases by a modified nucleoside
requires its activation by cellular nucleoside kinases and conversion
into a corresponding nucleoside triphosphate. Administration of AZT
phosphoramidate 9 as a substitute for AZT nucleoside triphosphate can
therefore eliminate a requirement for kinase activation. However, it is
important to assess the ability of HIV RT to recognize and insert AZT
phosphoramidate 9 with satisfactory efficiency. A potential drawback of
this approach could be the charged nature of aspartyl phosphoramidate
nucleotides. As a charged molecule, the aspartyl phosphoramidate of AZT
is not likely to pass through a cellular membrane unless active transport
is involved. However, intracellular diffusion is likely facilitated by
masking the negative charges of carboxylate moieties by means of
esterification as shown in FIG. 2B. Once a protected amino acid
phosphoramidate analogue is in the cytosol, it can be transformed back to
a charged, acidic form through the action of cellular esterases. These
principles of monophosphate activation and subsequent inhibition of viral
polymerases, as shown in FIGS. 2A and 2B for AZT, equally apply to other
chain-terminating nucleotides known in the art.

Manufacture of the Compounds of the Invention

[0239] The synthesis of amino acid phosphoramidate nucleotide analogues of
this invention may be accomplished according to the method illustrated by
scheme 2, starting from a nucleoside monophosphate, which itself can be
tailor-made by phosphorylation of a suitable nucleoside.

##STR00014##

[0240] The compounds according to the invention may be synthesized by
derivatisation of the 5'-mono-phosphate nucleoside precursor molecule as
illustrated in Scheme 2. In a first step (a), the phosphate group of the
5'-mono-phosphate nucleoside is coupled with the Z-group of the reagent
represented by the structural formula (E)

##STR00015##

wherein Z, R4 and R5 are the same as defined in the structural
formula (IV). Said coupling reaction results in the formation of a
phosphate ester (when Z=O), phosphate amide (when Z=NH or N--R) or
phosphate thioester (when Z=S). Said coupling reaction may be performed
using any coupling agent (also referred to as dehydrating agent) known in
the art for esterification or amide formation, in particular using a
carbodiimide coupling agent, more particularly dicyclohexylcarbodiimide
(DCC). The coupling reaction is preferably performed at a temperature
between room temperature and reflux temperature of the solvent. Depending
upon the nature of R4 and R5, additional acid, hydroxy or amine
functionalities in reagent (E) may be transiently protected to prevent
these functionalities from interfering with the condensation reaction
between the phosphate acid and Z. Therefore, the synthetic route may
provide for an optional subsequent step of deprotecting such
functionalities.

[0241] Schemes 3 and 4 below illustrate synthetic routes for the synthesis
of pyrimidine and purine derived compounds according to the present
invention respectively.

##STR00016##

##STR00017##

[0242] Alternatively, the compounds according to the structural formula
(I), or any specific embodiments thereof, may be obtained using a
synthetic procedure as illustrated by scheme 1 herein above.

[0243] The following examples are provided for illustrative purpose only,
and should not be considered as limiting the scope of the present
invention.

EXAMPLE 1

Synthesis

[0244] Scheme 5 below depicts 16 specific phosphate-modified nucleosides,
but is applicable to other phosphate-modified nucleosides disclosed in
this invention by introducing other substituents for R1 and R2
(i.e. using other alkyl residues instead of methyl). L-amino acids were
used for synthesis of all phosphoramidate analogues of the examples
wherein said amino acid is coupled to the phosphor atom with its
α-amino function. However, this procedure is applicable to all D-
and L-amino acids (both natural and unnatural) and organic
phosphate-modified nucleotides that contain an amino group. The
deprotection of the amino acid moiety was carried out with 0.4 M sodium
hydroxide in methanol-water solution. For the isolation of the pure
analogue phosphate-modified nucleotides, silica gel column chromatography
was employed with iPrOH--NH3--H2O mixture as an elution system.
A series of phosphoramidate analogues coupled to a variety of natural
L-amino acids synthesized as examples in this study is shown in Scheme 5
and FIG. 1. Step (a) of the synthesis illustrated by scheme 5 was
performed with the use of DCC as a coupling agent in a mixture of water
and tert-butanol under reflux temperature. Step (b) illustrates an
optional saponification using 0.4M NaOH in a mixture of water and
methanol.

[0245] A typical experimental procedure for the synthesis of 5'-amino acid
phosphoramidates of 2'-deoxyadenine, which is also generally applicable
to other phosphoramidates of the invention, is presented below.

[0247] Standard mass spectra (MS) were measured with a Finnigan LCQ DuO
(Thermo Fischer Scientific) using the ionization by electron impact
technique, data were acquired with the LAC/E32 system (Waters).

[0248] Exact mass spectra (MS) were obtained with a Q-T of 2®
(Micromass Ltd.) coupled to a CapLC® system (Waters).

[0249] Chemicals of analytical or synthetic grade were obtained from
commercial sources (dAMP: Sigma Aldrich; DCC: Fluke; thionyl chloride,
phthalic acids and triethylamine: Acros) and were used as received.

[0250] Dimethyl esters of dicarboxylic acids were either obtained from
commercial sources or prepared according to standard procedures.

[0312] A typical single nucleotide incorporation and primer extension
assays using HIV RT polymerase was carried out similar to one described
in the literature. HIV RT polymerase was purchased from GE Healthcare and
was supplied as either 12 or 30 U/μL stock solution. The final
concentration of HIV RT in a reaction mixture was 0.03-0.003 U/μL. A
primer/template duplex mixture was prepared by combining appropriate
amounts of a 5'-33P-labeled primer (P1) and a template in 1:2 molar
ratio to provide a 4× solution with 0.5 μM primer concentration.
The polymerase reactions were carried out in a 5×RT buffer
containing 250 mM TRIS.HCL, 250 mM KCl, 50 mM MgCl2, 2.5 mM
spermidine, 50 mM DTT, pH 8.3. For the single nucleotide incorporation
assay, a P1/T1 primer-template pair was used whereas the primer extension
study was performed with P1/T3 and P1/T5 primer-template pairs.

[0313] Briefly, a mixture containing 5 μL of the 4× primer/DNA
duplex solution and 5 μL of 4× polymerase solution was
pre-incubated at 37° C. for 2 minutes to ensure formation a DNA
polymerase/DNA duplex complex. Simultaneously, 2× solutions of dNTP
to provide final concentrations of 100 μM, 200 μM, 500 μM and 1
mM were also pre-incubated at 37° C. for 2 minutes as well. The
polymerase reaction in a total volume of 20 μL was initiated by
addition of 10 μL of the 2× dNTP solution to the DNA
polymerase/DNA duplex mixture. The reactions were run at 37° C.
and aliquots (2.5 μL) were removed at specified time points and mixed
with 10 μL of a quenching buffer containing 80% formamide, 2 mM EDTA,
1×TBE. Quenched reactions were analyzed by loading 2 μL of a
reaction sample onto a 20% denaturing polyacrylamide:gel (19:1
acrylamide:bisacrylamide, 7M urea, 0.4 mm×30 cm×40 cm) made
in 1×TBE buffer (90 mM Tris-borate, 2 mM EDTA, pH 8.3). The gel was
pre-equilibrated for 2 hours before loading and the reactions were run
along with a tracking dye marker containing bromophenol blue (BB) and
xylene cyanol (XC) until the BB dye had run 2/3 of the total gel length.
For visualization, gels were scanned using a Cyclone Phosphoroimager.

[0314] Primer extension study with HIV RT and Therminator DNA polymerase
was performed in a fashion similar to the single nucleotide incorporation
experiments. Two sets of primer/template complexes were used: P1/T3 and
P1/T5.

Single Nucleotide Incorporation by Therminator DNA Polymerase

[0315] In the case of Therminator DNA polymerase, the enzyme was obtained
from Westburg (NEB) (2 U/μL) and the reactions were carried out in a
10× Thermopol reaction buffer containing 20 mM TRIS.HCl, 10 mM KCl,
2 mM MgSO4, 0.1% Triton X-100, pH 8.8. The final concentration of
the Therminator DNA pol in the reaction mixture was 8.33×10-4
U/μL. The polymerase reaction involving Therminator DNA polymerase or
any other thermostable polymerase (Vent (exo.sup.-), Taq DNA polymerase)
was carried out in a similar way as the HIV RT reaction with some
modifications. The dNTP solutions and the primer/template/DNA polymerase
mixture solutions were topped with mineral oil (30-60 μL) and
pre-incubated at 70° C. for 2 minutes. The polymerase reactions
were performed at 70° C. as well.

Steady State Kinetics of Single Nucleotide Incorporation

[0316] The steady steate kinetics of a single nucleotide incorporation of
an amino acid phosphoramidate (AA-dAMP) and of a natural nucleoside
triphosphate (dATP) was determined by the gel-based polymerase assay. In
all the experiments, the template T1 and the primer P1 were used (Table
1). The primer and template in 1:2 molar ratio were hybridized in a
buffer containing 20 mM TRIS.HCl, 10 mM KCl, 2 mM MgSO4, 0.1% Triton
X-100, pH 8.8 and used in an amount to provide 125 nM concentration of
the primer in each 12 μL reaction. The range of concentrations for
amino acid phosphoramidates was optimized according to a Km value
for the incorporation of an individual nucleotide. In the case of HIV RT
(Amersham Bioscience (GE Healthcare), 30 U/μL), reaction mixtures
containing the enzyme in concentration to attain 5-20% conversion and
appropriate substrate concentration (nucleoside phosphoramidate or
natural dNTP) were incubated at 37° C. and run for 8-10 different
time intervals. Kinetic analysis of single nucleotide incorporation by
Therminator DNA polymerase (NEB, 2 U/μL) was performed similarly
except the incubations and polymerase reactions, which were carried out
at 70° C. To prevent solvent condensation/evaporation, the
reaction mixtures were topped with mineral oil. The reactions were
quenched by addition of the buffer containing 80% formamide, 2 mM EDTA,
1×TBE buffer. The analysis of polymerase reactions was performed by
polyacrylamide gel electrophoresis (20% (19:1 mono:bis), 7M urea, 30
cm×40 cm×0.4 mm). The relative band intensities were measured
using OptiQuant software. The rates of incorporation (V) were calculated
based on the percentage of the extension production (n+1 band). The
kinetic parameters (Vmax and Km) were determined by plotting V
(pmol/minU) versus substrate concentration and fitting the data point to
a rectangular hyperbola using GraphPad Prism software.

EXAMPLE 3

Synthesis of Oligonucleotides

Single Nucleotide Incorporation by HIV RT

[0317] HIV reverse transcriptase is involved in the copying of the HIV
genome and uses deoxy- and ribonucleotides as substrates. Furthermore,
HIV RT is an error-prone polymerase and has high mutation rate.
Therefore, the essential role of the HIV RT in viral replication and its
flexibility and tolerance toward modified nucleotides renders this enzyme
a primary target in treatment of HIV infection. In the presented study,
the ability of HIV RT to incorporate a series of amino acid
phosphoramidate nucleoside analogs was investigated by the gel-based
single nucleotide incorporation assay.

[0319] This phosphoramidate analog was recognized by HIV RT and
efficiently incorporated into a growing primer strand resulting in 90%
conversion to a (n+1) strand in 60 min (500 μM nucleotide
concentration). At the same conditions, incorporation of His-dAMP(6),
Gly-dAMP (3), and Pro-dAMP (8) was 1.5, 6.5, and 3.7 fold less efficient,
respectively. Efficient incorporation of Asp-dAMP (24.1%) was also
observed when the substrate concentration was decreased 10 fold. However,
significantly lesser incorporation of amino acid phosphoramidate was
detected for nucleosides coupled to non-polar, hydrophobic amino acids.
Ala-dAMP and Tyr-dAMP behaved as poor substrates leading to merely 7- and
10-fold reduction in primer extension, respectively (FIG. 4).

[0320] Interestingly, no incorporation occurred when respective methyl
ester derivatives 1a-8a were used as substrates in the polymerase
reaction. Another unexpected result was observed with Glu-dAMP analog (2)
that also acted very poorly as a HIV RT substrate. These observations
suggest that recognition and incorporation of AA dAMPs are likely to be
dictated by the chemical structure and electrostatics of the amino acid
moiety.

Single Nucleotide Incorporation by Therminator DNA Polymerase

[0321] Yet another polymerase enzyme that demonstrates similar trends in
recognition and utilization of AA-dAMPs is Therminator DNA polymerase, a
variant of (9° N-7) Thermococcus sp. DNA polymerase. This enzyme
demonstrated effective recognition and incorporation of a number of
nucleotides bearing unnatural nucleobase and sugar moieties. Likewise,
probing of AA-dAMP incorporation directed by Therminator DNA polymerase
revealed property of analogs 1, 3, and 6 to act effectively as
alternative substrates in the DNA polymerization reaction (FIGS. 5 and
6).

[0322] Yet again, the best results were obtained with Asp-dAMP, which led
to 25.2% primer extension over 60 min at 500 μM nucleoside
concentration. At the same conditions, the similar results were obtained
for Gly-dAMP and His-dAMP (26% and 25.4% primer extension, respectively).
In the case of Glu-dAMP and methyl protected AA-dAMPs, Therminator DNA
polymerase displays selectivity analogous to HIV reverse transcriptase
and fails to direct incorporation of those phosphoramidate analogs (FIG.
6).

Single Nucleotide Incorporation by Other DNA Polymerases

[0323] The remarkable property of Asp-dAMP encouraged further
investigation and testing 1 as a substrate for other DNA polymerases.
However, in the case of Taq, Vent (exo.sup.-), and KF (exo.sup.-) DNA
polymerases, recognition and incorporation efficiency were significantly
less appealing. Incorporation and primer extension were observed only in
the case of KF (exo.sup.-) DNA pol demonstrating 32.5% conversion of the
primer strand in 60 minutes. This is in contrast to Taq and Vent
(exo.sup.-) DNA polymerases that failed to insert 1 into a growing primer
strand. The diversity in incorporation selectivity that are observed
among the polymerases (Therminator, Taq, Vent (exo.sup.-), KF
(exo.sup.-), and HIV reverse transcriptase) could indicate the
differences in the active site flexibility and tolerance to the
triphosphate modifications (FIG. 7).

Primer Extension by HIV RT

[0324] The further investigation of Asp-dAMP recognition by the reverse
transcriptase focused on ability of HIV RT to direct template dependent
incorporation of more than one phosphoramidate nucleosides. For this
purpose, template T3 containing a string of three thymidine nucleobases
flanked with cytidine nucleobases at the 3' end and the template T5 that
has an overhang of seven thymidine residues were used. Ability to HIV RT
to synthesize a DNA sequence using phosphoramidate nucleotides as
substrates was tested among Asp-dAMP, His-dAMP, Gly-dAMP, and Pro-dAMP.

[0325] Among this series of phosphoramidate nucleoside, the most
encouraging results using T3 template were observed with Asp-dAMP and
His-dAMP which were used by HIV RT to extend a primer with three adenine
nucleobases (n+3 product) (FIGS. 8 and 9).

[0326] However, after 60 minutes of the polymerase reaction the (n+2)
product predominates over the (n+3) product (56.3% vs. 5.2% for Asp-dAMP
and 67.1% vs. 13.5% for His-dAMP). Interestingly, efficiency of DNA
synthesis with His-dAMP at 500 μM substrate concentration is similar
or better to that when Asp-dAMP serves as the substrate (67.1% vs. 56.3%,
respectively, for the synthesis of the (n+2) primer). This is in contrast
to the single nucleotide incorporation results that indicate that
His-dAMP is worse than Asp-dAMP as a substrate for HIV RT.

[0327] In the case of the T5 template with the overhang of seven thymidine
nucleobases, HIV RT indeed generates (n+6) and (n+7) products at a very
little extent while the (n+2) and (n+3) products are prevalent (FIG. 10).
The obvious stalling of the HIV RT polymerase after incorporation of two
adenine nucleobases might indicate substrate inhibition or a template
sequence effect. The primer strand extension for one hour with 500 μM
of Gly-dAMP or Pro-dAMP takes place with low efficiency and does not
result in the formation of the full-length extension products.

Primer Extension by Therminator DNA Polymerase

[0328] The Therminator DNA pol mediated addition of amino acid
phosphoramidate nucleosides instead of natural dNTPs at the 3' terminal
end was investigated for several AA-dAMPs. Similarly, to the case of HIV
RT, the best results were observed with AspdAMP phosphoramidate, which
was successfully incorporated across from a string of thymidine residues
(T3 template) to provide a (n+3) product (FIG. 11). However, when
His-dAMP and Gly-dAMP were used as substrates for Therminator DNA
polymerase, the primer extension took place with significantly lesser
efficiency (13.6% and 18.1% of primer extension, respectively) with the
(n+1) product being predominant and halted after addition of two
nucleoside phosphoramidate residues (FIG. 12). The primer extension with
Pro-dAMP was very ineffective and resulted only in addition of one
nucleoside phosphoramidate residue at the primer's end.

[0329] In the case of the T5 template with the overhang of seven thymidine
residues, the predominant product of the primer extension was the (n+2)
oligonucleotide. Nonetheless, Therminator DNA polymerase was able to
carry out the extension of the T5 primer with Asp-dAMP phosphoramidate
and incorporate up to five adenine residues.

HIV Reverse Transcriptase

[0330] Steady state kinetics for single nucleotide incorporation was
determined by the gel based polymerase assay. The kinetics analysis
(Table 1) shows that Vmax for incorporation of 1 is only 3 fold
lower than that for a HIV RT natural substrate (dATP), however, the
Km value for Asp-dAMP is 400 fold higher as compared to dATP.
Therefore, as a result, the specificity, or a Vmax/Km value,
for insertion of amino acid phosphoramidate against a natural nucleobase
is reduced 1300 fold. The significantly higher Km value for an
unnatural amino acid phosphoramidate analog than one for the natural
substrate suggests that the phosphoramidate substrate dissociates from
the active site more readily and faster. However, small difference in
Vmax values (3 fold) indicates that once the amino acid
phosphoramidate substrate is bound at the active site nucleotidyl
transfer takes place with the efficiency and the rate slightly lower than
in the case of the natural substrate. It probably also indicates that the
bound Asp-dAMP fits the polymerase active site well and adapts geometry
and orientation that closely resembles dATP.

[0332] 2'-Deoxynucleoside 5'-monophosphate (1.00 mmol) and L-aspartic acid
methyl ester/N-Me-L-aspartic acid methyl ester (7.00 mmol) were dissolved
in tBuOH (9.1 mL) and water (3.1 mL). A few drops of triethylamine
were added to facilitate dissolution. Then, a solution of DCC (5.00 mmol)
in tBuOH (6.7 mL) was added and the reaction mixture was refluxed
for 2-3 hours while stirring under argon. The progress of the reaction
was followed by TLC using a iPrOH:NH3:H2O (7:1:2) mixture. Upon
completion, the reaction mixture was cooled down and the solvents were
removed by rotatory evaporation. The residue was resuspended in water and
extracted with diethyl ether. The aqueous phase was then lyophilized. The
residue was subjected to column chromatography on silica gel using the
following solvent gradient: CHCl3:MeOH (5:1),
CHCl3:MeOH:H2O (5:2:0.25), CHCl3:MeOH:H2O (5:3:0.5),
and finally CHCl3:MeOH:H2O (5:4:1). The product obtained was
treated with 1.4 M K2CO3 (20 eq.) in MeOH:H2O (1:2) and
the reaction mixture was allowed to stir at room temperature. The course
of the reaction was monitored by TLC using a iPrOH:NH3:H2O
(6:3:1) mixture until disappearance of the starting material. Then, the
solvents were removed under reduced pressure. The residue was subjected
to column chromatography on silica gel using the following solvent
gradient: iPrOH, iPrOH:NH3:H2O (7:1:1).

[0354] Oligodeoxyribonucleotides P1, P2, T1, T2, T4 and A were purchased
from Sigma Genosys. The concentrations were determined using a Varian
Cary 300 Bio UV-spectrophotometer.

[0355] The lyophilized oligonucleotides were dissolved in DEPC-treated
water and stored at ±20° C. The primer oligonucleotides were
5'-33P-labeled with 5'-[γ33P]ATP (GE Healthcare) using T4
Polynucleotide kinase (NEB) following standard procedures. Labeled
oligonucleotides were further purified using Illustra® Microspin®
G-25 columns (GE Healthcare).

DNA Polymerase Reactions

[0356] End-labeled primers were annealed to their template by combining
primer and templates in a molar ratio of 1:2 and heating the mixture to
70° C. for 10 minutes, followed by slow cooling to room
temperature over a period of 2.5 hours. For the incorporation of
L-Asp-dGMP (9), L-Asp-dTMP (10), L-Asp-dCMP (11), and N-Me-Asp-dAMP (12)
(see FIG. 13), the primer P1 was annealed to templates T1, T2, T4 or A
and P2 was annealed to T4. A series of 20 μL reactions was performed
for the enzyme HIV reverse transcriptase (GE Healthcare, 30 U/4 stock
solution). The final mixture contained 125 nM primer-template complex, RT
buffer (250 mM TrisHCl, 250 mM KCl, 50 mM MgCl2, 2.5 mM spermidine,
50 mM DTT, pH 8.3), 0.03 U/μL HIV RT and different amino acid
phosphoramidates building blocks concentrations. In the control reaction
with the natural nucleotide, 10 μM or 50 μM dGTP, dTTP, dATP or
dCTP were used. The mixture was incubated at 37° C. for 3 minutes
and aliquots were quenched after 5, 10, 20, 30, 60 and 120 minutes
respectively.

Kinetics Experiments

[0357] To determine the kinetic parameters of the incorporation of
Asp-dGMP (9), Asp-dTMP (10), Asp-dCMP (11) or dGTP, dTTP, dCTP, a
steady-state kinetic assay was carried out. The reaction mixture was
started by adding HIV reverse transcriptase to P1-T2, P1-T4 or P2T4
complex, buffer and Asp-dGMP, Asp-dTMP, Asp-dCMP or dGTP, dTTP, dCTP. The
final mixture (20 μL) contained 0.015 or 0.0075 U/μL HIV reverse
transcriptase, buffer, 125 nM primer-template complex and various
concentrations of Asp-dNMP or dNTP. A concentration range of 0.01-1 mM
was used for Asp-dNPM and a concentration range of 0.1-10 μM was used
for dNTP. Reactions were incubated at 37° C. Reaction times were
between 1 and 120 minutes. The kinetic constants Vmax and KM
were determined from a Michaelis-Menten plot, using GraphPad Prism
version 3.02.

Electrophoresis

[0358] All polymerase reactions (3 μL) were quenched by the addition of
10 μL of loading buffer (90% formamide, 0.05% bromophenol blue, 0.05%
xylene cyanol and 50 mM EDTA). Samples were heated at 70° C. for 5
minutes prior to analysis by electrophoresis for 2-3 hours at 2000 V on a
0.4 mm 20% denaturing gel in the presence of a 100 mM Tris-borate, 2.5 mM
EDTA buffer, pH 8.3. Products were visualized by phosphor imaging. The
amount of radioactivity in the bands corresponding to the products of
enzymatic reactions was determined by using the Optiquant image analysis
software (Perkin Elmer).

[0359] In this example 4, we demonstrate that the L-aspartyl (1) and the
L-histidyl (6) derivatives of dAMP can function as a substrate for human
immunodeficiency virus reverse transcriptase type 1 (HIV-RT) and that
chain elongation is possible. These experiments demonstrate that the
polymerase enzyme is able to catalyse the cleavage of the relatively
stable P--N bond and that an amino acid can be used as leaving group
during the polymerization reaction.

[0360] In order to further characterize the potentiality of this leaving
group, we studied the substrate properties L-Asp-dGMP(9), L-Asp-dTMP(10)
and L-Asp-dCMP(11) for HIV-RT. Thus, the present work focuses on the
ability of HIV RT to incorporate aspartic acid derivatives of
2'-deoxyguanosine-5'-monophosphate, 2'-deoxythymidine-5'-monophosphate,
and 2'-deoxycytidine-5'-monophosphate into a growing DNA chain.

[0361] Kinetic analysis of L-Asp-dAMP (1) incorporation (in example 3)
showed that the incorporation efficiency of this substrate by HIV RT was
lower than that for the natural substrate (dATP). Therefore, we
considered the possibility of activating the nucleoside monophosphate
with modified aspartic acid derivatives. In this regard, we focused first
attention on N-methyl-L-aspartic acid as leaving group. Thus,
2'-deoxyadenosine-5'-N-methylaspartyl phosphoramidate (12) was
synthesized from 2'-deoxyadenosine-5'-monophosphate and
N-methyl-L-aspartic acid methyl esther according a method well known in
the art.

[0362] The ability of HIV RT to incorporate 5'-N-methylaspartyl
phosphoramidate 12 into a growing DNA chain was explored in a first
series of gel-based single-nucleotide-incorporation assays with primer P1
and template T1 (FIG. 14, panel I). Compound 12 is incorporated into the
growing DNA strand but without base-pairing selectivity, since it is
easily misincorporated opposite a cytosine base to form the corresponding
n+2 product. The same infidelity in incorporation profile was found using
lower polymerase concentration. The behavior of 12 is in contrast with
that of the L-aspartic acid derivative 1, which inserted selectively
against thymine to give the n+1 product (as shown in example 3). When we
carried out a control experiment with 12 and a mismatch sequence (A
against A, P1T4, see FIG. 15, panel I), phosphoramidate 12
misincorporated against adenine. Compound 12 is a good substrate for
HIV-RT, but it shows reduced selectivity compared to other L-aspartic
acid derivatives in these experiments. However, the incorporation of this
compound 12 resembles most the properties of the natural substrate
(dATP); thus compounds like 2'-deoxyadenosine-5'-N-methylaspartyl
phosphoramidate (12) could be the best phosphate-modified nucleosides of
the invention.

[0363] L-Asp-dGMP (9), L-Asp-dTMP (10), and L-Asp-dCMP (11) were
synthesized according to a previously reported method. A series of
gel-based single-nucleotide-incorporation assays was carried out (FIG.
14), by using primer P1 and template T2 for guanine (9, Panel II), P1 and
T4 for thymine (10, Panel III), and P2 and T4 for cytidine (11, Panel
IV). As shown before for L-Asp-dAMP (1) in example 3, these three
phosphoramidate analogues are recognized by HIV RT and are efficiently
incorporated with base-pairing selectivity into the corresponding growing
strand, resulting in 92% (9), 95% (10), and 82% (11) conversion to a n+1
strand in 60 min (500 μM nucleotide concentration). It is interesting
to note that no incorporation was observed when HIV RT was replaced for
M-MLV reverse transcriptase, AMV reverse transcriptase and Φ-DNA
polymerase.

[0364] In order to confirm that the former observations were due to a true
base-pair extension instead to an extendase/terminal transferase activity
we tried to extend a blunt ended duplex. With this purpose we carried out
a third series of gel-based single-nucleotide-incorporation experiments
with primer P1, template A (see FIG. 15) and phosphoramidates 1, 9, 10
and 11. As expected, none of these compounds incorporated at all into the
growing DNA strand. Furthermore, control experiments with a mismatch
sequence (G against A, P1T4, 9; T against C, P1T2, 10; C against T, P1T1,
11; see FIG. 15) confirmed a true base-pair extension, since primer
elongation was not observed.

[0365] The efficiency of incorporation by HIV RT of compounds 9, 10, and
11 was investigated by determination of the kinetic parameters KM
and Vmax. As observed for 1 before (as described in example 3),
steady-state kinetic analysis (Table 3) of 9, 10 and 11 incorporation
indicated that, although KM for the aspartic acid phosphoramidate
analogues is significantly higher than for the natural substrates, the
measured Vmax is only 9-13 fold lower. These data suggest fast and
efficient nucleophilic displacement of the amino acid moiety once the
aspartic acid phosphoramidate is bound at the active site.

[0366] Next, we analyzed the ability of HIV RT to direct template
dependent selective incorporation of more than one different
phosphoramidate nucleosides. For this purpose, primer P1 and template T2
containing an overhang of one cytidine and four thymine nucleobases at
the 3' end were used. The ability of HIV RT to synthesize a DNA sequence
with L-Asp-dGMP (9) and L-Asp-dAMP (1) was tested (FIG. 16). We carried
out first the incorporation of 9 into the growing primer DNA strand.
After complete conversion to the n+1 product (two hours), compound 1 was
added and the reaction was followed for two more hours. At this time, the
n+2 product predominated over the n+1 product (80% vs 20%). In these
experiments, stalling of the HIV RT appeared after incorporation of two
nucleotides. To confirm that the n+2 product formation is due to adenine
incorporation instead of guanine misincorporation a control experiment
was carried out. Primer P1, template T2 and compound 9 were used and the
reaction was followed for four hours. As expected, no misincorporation
was observed, the n+1 conversion at 4 hours is the same as observed at 2
hours (95% incorporation).

EXAMPLE 5

Synthesis of Phosphoramidates and Phosphodiesters

[0367] The synthesis of the methyl esters of the phosphate-modified
nucleosides of the invention was accomplished according to the method
described by Wagner et al. in Mini-Rev. Med. Chem. (2004) 4:409, starting
from a nucleoside monophosphate. Deprotection of the methyl esters was
carried out with potassium carbonate in methanol-water solution.

[0368] The synthesis of compounds 22-24, the structural representation of
which is shown in FIG. 17, was carried out in two steps involving the
production of ester intermediates according to the following detailed
procedure.

[0399] HIV-1 Reverse Transcriptase serves, in the HIV-1 viral replication
process, as a catalyst and uses deoxynucleotides as substrates. This
polymerase is error-prone and thus has a high mutation rate. Here, we
evaluated the capacity to incorporate a deoxyadenosine nucleoside into
the primer-template complex P1T1 using HIV-1 RT, and some of the above
described illustrative compounds of the invention, carrying different
leaving groups, as substrates. The initial screening was carried out
using a template with an overhang of one thymidine nucleotide followed by
three non-pyrimidine bases (Table 1). Incorporation efficiency was
analysed by the polyacrylamide gel-based single nucleotide incorporation
assay.

[0400] The isophthalic acid-derived phosphodiester (22) was recognized by
HIV-1 RT and efficiently incorporated into a growing primer strand (as
shown in FIG. 18) with a conversion to an n+1 strand 90-92% (22) over a
period of 2 hours at 1 mM concentration. The corresponding
anilino-derived phosphate nucleoside (23) was less well recognized as
substrate. Finally, little incorporation (13% n+1 product after 2 hours)
was observed with the phthalic acid dAMP derivative (24).

[0401] A few interesting observations can be drawn from this first panel.
Despite the geometric constraint brought by the aromatic ring,
dicarboxylated phenol and dicarboxylated aniline can still function as
leaving groups in a polymerase catalyzed reaction. A phenolate is a
better leaving group than the corresponding aniline anion, although it is
unclear whether protonation of the nitrogen atom of the anilino group may
be involved in the catalytic mechanism.

[0402] Among the two substituted phenol moieties, the one carrying both
carboxyl substituents in meta position (22) is more successful than the
one carrying the carboxyl substituents in meta and para positions
respectively (24). This indicates that the orientation of both carboxyl
functions is important, which might be attributed to steric hindrance in
the active site of the polymerase, or to more specific chelating
properties. Compound (22) was further evaluated at different
concentrations (as shown in FIG. 19). At 500 μM compound 2 displayed
75% of n+1 formation, which represents 88% of L-Asp-dAMP capacity.

[0403] We also tested the possibility of a polymerase independent
incorporation, but no compound was incorporated in the absence of the
enzyme.

EXAMPLE 7

[0404] Oligodeoxyribonucleotides P1, T1, T2 and T3 were purchased from
Sigma Genosys. The concentrations were determined with a Varian
Cary-300-Bio UV Spectrophotometer.

[0405] The lyophilized oligonucleotides were dissolved in
diethylpyrocarbonate (DEPC)-treated water and stored at -20° C.
The primer oligonucleotides were 5'-33P-labeled with
5'-[γ33P]-ATP (Perkin Elmer) using T4 polynucleotide kinase
(New England Biolabs) according to standard procedures. The labeled
oligonucleotide was further purified using Illustra® Microspin®
G-25 columns (GE Healthcare).

DNA Polymerase Reactions

[0406] End-labeled primer was annealed to its template by combining primer
and template in a molar ratio of 1:2 and heating the mixture to
70° C. for 10 minutes followed by slow cooling to room temperature
over a period of 1.5 hour. For the incorporation of 1, 2, 3, 4, 5 and 6,
a series of 20 μL-batch reactions was performed with the enzyme HIV-1
RT (Ambion, 10 U/μL stock solution, specific activity 8.095 U/mg,
concentration 1.2 mg/mL). The final mixture contained 125 nM primer
template complex, RT buffer (250 mM Tris.HCl, 250 mM KCl, 50 mM
MgCl2, 2.5 mM spermidine, 50 mM dithiothreitol (DTT); pH 8.3), 0.025
U/μL HIV-1 RT, and different concentrations of phosphoramidate or
phosphodiester building blocks (1 mM, 500 μM, 200 μM and 100 μM
respectively). In the case of the aromatic phosphate-modified nucleosides
23 and 24, the range of concentrations was limited to 1 mM. In the
control reaction with the natural nucleotide, a 10 μM dATP
concentration was used. The mixture was incubated at 37° C. and
2.5 μL aliquots were quenched after 5, 10, 20, 30, 60 and 120 minutes.
Results are shown in FIG. 18.

EXAMPLE 8

[0407] In an attempt to further simplify the structure of the leaving
group, we investigated the pyrophosphate mimicking ability of a group
carrying only one carboxylic acid unit, for both the phosphoramidate
nucleoside β-Ala-dAMP (a) and the phosphodiester nucleoside glycolic
acid-dAMP (b) structurally shown below.

##STR00023##

Synthesis of Phosphoramidates and Phosphodiesters

[0408] For the synthesis of glycolic acid dAMP (b), a method of divalent
cation assisted coupling, as shown in the scheme below (wherein NEM
stands for N-ethyl morpholine) and previously suggested by Sawai in Bull.
Chem. Soc. (1990) 63:692-696, was used. In this approach, the carboxyl
acid moiety does not need to be protected since it serves as a ligand for
the divalent metal-ion during the nucleotidyl transfer, thus requiring
one single synthetic step. The desired phosphodiester nucleoside was
obtained in a 41% yield (after high performance liquid chromatography
purification).

##STR00024##

EXAMPLE 9

Single Incorporation

[0409] Using the glycolic acid dAMP phosphodiester (b) of example 8
(GA-dAMP), the incorporation efficiency into P1T1 by HIV-1 reverse
Transcriptase was moderate, as n+1 formation was observed up to 68% at a
concentration of 500 μM as shown in FIG. 20. This shows that, in the
case of a phosphodiester bond between the nucleoside monophosphate and
the leaving group, one carboxylic acid function on the leaving group is
enough to enable the nucleotidyl transfer reaction.

[0423] All publications, patents, and patent applications mentioned in
this specification are herein incorporated by reference to the same
extent as if each independent publication or patent application was
specifically and individually indicated to be incorporated by reference.

[0424] While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modifications and this application is intended to cover any variations,
uses, or adaptations of the invention following, in general, the
principles of the invention and including such departures from the
present disclosure that come within known or customary practice within
the art to which the invention pertains and may be applied to the
essential features hereinbefore set forth, and follows in the scope of
the claims.